Partial Region Polypeptide of REIC/Dkk-3 Protein

ABSTRACT

A polypeptide capable of strongly inducing and activating dendritic-cell-like cells for treating or prevent cancer by immunotherapy, and DNA encoding the polypeptide. The polypeptide is a polypeptide (a) or (b) consisting of a partial region of the REIC/Dkk-3 protein.

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition fortreating or preventing cancer, which enhances anticancer immuneactivity.

BACKGROUND ART

The REIC/Dkk-3 gene is known to be associated with cell immortalization,and suppression of expression of such genes in cancer cells has beenreported (International Publication WO01/038523 pamphlet, Tsuji, T. etal., Biochem Biophys Res Commun 268, 20-4 (2000), Tsuji, T. et al.,Biochem Biophys Res Commun 289, 257-63 (2001), Nozaki, I. et al., Int JOncol 19, 117-21 (2001) and Kurose, K. et al., J Urol 171, 1314-8(2004)).

The REIC/Dkk-3 gene is a member of the Dkk family, and it is suggestedthat such gene inhibits Wnt signal transduction via a Wnt receptor(Bafico, A. et al., Nat Cell Biol 3, 683-6 (2001) and Hoang, B. H. etal., Cancer Res 64, 2734-9 (2004)). It is reported that the Wnt geneplays multiple roles in important biological conditions, such as cellgrowth, differentiation, and canceration (Moon, R. T. et al., Science296, 1644-6 (2002)).

It has been reported concerning REIC/Dkk-3 that when the full-lengthREIC/Dkk-3 protein is added at a concentration of 10 μg/ml to a culturesolution in which peripheral blood mononuclear cells (monocytes) arecultured, the cells differentiate into dendritic-cell-like cells(International Publication WO09/119,874 pamphlet).

In general, there are only a small number of references that disclosesubstances recognized as having the ability to induce dendriticcell(-like) differentiation from blood precursor cells. Most suchsubstances are well-known cytokines. For instance, there are manyreports on the combined use of GM-CSF and IL-4; that is, the inductionof dendritic cell differentiation from monocytes through the addition ofGM-CSF and IL-4 to the culture solution. This combination is called the“gold standard” for dendritic cell differentiation. In addition, assubstances capable of inducing dendritic cell differentiation when usedalone or in combination, TNF-alpha, IL-2, IL-3, IL-6, IL-7, IL-12,IL-13, IL-15, HGF (hepatocyte growth factor), a CD40 ligand, M-CSF, anFit3 ligand, and TGF-β have been reported. Among these proteins, knownexamples of a substance capable of inducing precursor cells todifferentiate into dendritic cell(-like) cells when used alone includeIL-2, IL-15, HGF, and a CD40 ligand, as in the case of the full-lengthREIC/Dkk-3 protein. Of these, only IL-2 has been confirmed to have invivo anticancer effects. A possible reason why GM-CSF is unable toexhibit such effects is that it induces not only anticancer immunity,but also the differentiation of immunosuppressive cells represented bybone marrow-derived immunosuppressive cells (myeloid-derived suppressorcells (MDSC)) or immunoregulatory T cells (Treg) so as to activate a“negative immune system” (Parmiani, G. et al., Annals of Oncology 18,226-32 (2007)).

SUMMARY OF THE INVENTION

An object of the present invention is to provide a polypeptide capableof strongly inducing and activating dendritic-cell-like cells, so as tobe able to treat or prevent cancer by immunotherapy, and DNA encodingthe polypeptide.

The present inventors have revealed the usability/superiority of afull-length REIC/Dkk-3 protein in in vivo immune/inflammatory phenomenaand the possible use thereof in wide-ranging fields and diseases(International Publication WO09/119,874 pamphlet).

The present inventors have further examined the activity of a partialregion of the REIC/Dkk-3 protein, and thus discovered that such aspecific partial region thereof has strong bioactivity to inducedendritic cell-like differentiation from monocytes and that thebioactivity is significantly higher than that of the full-lengthREIC/Dkk-3 protein. This demonstrates that the specific partial regionof the REIC/Dkk-3 protein induces dendritic-cell-like celldifferentiation from monocytes, and can be used for treating orpreventing cancer by activating anticancer immunity.

It has been reported that as such an immunological cancer therapeuticagent, an IL-2 protein may have therapeutic effects on specific types ofcancer such as renal cell carcinoma. However, it is a clinically knownfact that the types of cancer to which the IL-2 protein can beappropriately administered and the effects thereof are limited. It hasbeen confirmed that the REIC/Dkk-3 protein is expressed and secreted atdecreased levels in almost all types of cancer. Hence, the REIC/Dkk-3protein may be used as a cancer therapeutic agent having the effect ofactivating anticancer immunity for various types of cancer, and thetherapeutic effects thereof can be superior to those of IL-2.

As described above, the present inventors have discovered that aspecific partial region of the REIC/Dkk-3 protein has stronger“bioactivity to induce dendritic cell-like differentiation frommonocytes” than the full-length REIC/Dkk-3 protein, and thus they havecompleted the present invention.

Specifically, the present invention is as follows.

[1] A polypeptide that is any one of the following polypeptides (a) to(e), which consists of a partial region of a REIC/Dkk-3 protein:(a) a polypeptide consisting of a partial region of the REIC/Dkk-3protein, which consists of an amino acid sequence selected from thegroup consisting of

the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7 and SEQ ID NO: 9, and

the amino acid sequence of a polypeptide that contains the partialregion consisting of Gly at position 205 to Phe at position 288 of theamino acid sequence shown in SEQ ID NO: 2, and consists of a fragment ofthe partial region consisting of Ser at position 135 to Ile at position350 of the amino acid sequence shown in SEQ ID NO: 2;

(b) a polypeptide having activity of inducing dendritic-cell-like celldifferentiation from monocytes, which is a polypeptide proteinconsisting of an amino acid sequence that has a substitution, adeletion, or an addition of 1 or several amino acids with respect to anamino acid sequence selected from the group consisting of

the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7 and SEQ ID NO: 9, and

the amino acid sequence of a polypeptide that contains the partialregion consisting of Gly at position 205 to Phe at position 288 of theamino acid sequence shown in SEQ ID NO: 2, and consists of a fragment ofthe partial region consisting of Ser at position 135 to Ile at position350 of the amino acid sequence shown in SEQ ID NO: 2;

(c) a polypeptide consisting of a partial region of the REIC/Dkk-3protein, which can bind to a Tctex-1 protein and consists of the aminoacid sequence shown in SEQ ID NO: 17, and;(d) a polypeptide consisting of an amino acid sequence having asubstitution, a deletion, or an addition of 1 or several amino acidswith respect to the amino acid sequence shown in SEQ ID NO: 17 andhaving the activity of binding to the Tctex-1 protein; and(e) a polypeptide consisting of a partial region of the REIC/Dkk-3protein and being capable of binding to the Tctex-1 protein, whichcontains the consensus sequence shown in SEQ ID NO: 18 and consists of 7to 22 amino acid residues.[2] DNA that is any one of the following DNAs (f) to (l), which encodesa polypeptide consisting of a partial region of a REIC/Dkk-3 protein;(f) DNA consisting of a nucleotide sequence selected from the groupconsisting of

the nucleotide sequences shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8 and SEQ ID NO: 10, and

a nucleotide sequence that contains the partial sequence consisting of gat position 613 to c at position 864 of the nucleotide sequence shown inSEQ ID NO: 1, and consists of a fragment of the partial sequenceconsisting of t at position 403 to t at position 1050 of the nucleotidesequence shown in SEQ ID NO: 1;

(g) DNA hybridizing under stringent conditions to DNA consisting of anucleotide sequence complementary to a nucleotide sequence selected fromthe group consisting of

the nucleotide sequences shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8 and SEQ ID NO: 10, and

a nucleotide sequence that contains the partial sequence consisting of gat position 613 to c at position 864 of the nucleotide sequence shown inSEQ ID NO: 1, and consists of a fragment of the partial sequenceconsisting of t at position 403 to t at position 1050 of the nucleotidesequence shown in SEQ ID NO: 1, and

encoding a polypeptide having activity of inducing dendritic-cell-likecell differentiation from monocytes;(h) DNA consisting of a nucleotide sequence having a deletion, asubstitution, or an addition of 1 or several nucleotides with respect toa nucleotide sequence selected from the group consisting of

the nucleotide sequences shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8 and SEQ ID NO: 10, and

a nucleotide sequence that contains the partial sequence consisting of gat position 613 to c at position 864 of the nucleotide sequence shown inSEQ ID NO: 1, and consists of a fragment of the partial sequenceconsisting of t at position 403 to t at position 1050 of the nucleotidesequence shown in SEQ ID NO: 1, and

encoding a polypeptide having activity of inducing dendritic-cell-likecell differentiation from monocytes;(i) DNA consisting of a DNA sequence that encodes an amino acid sequenceselected from the group consisting of

the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7 and SEQ ID NO: 9, and

the amino acid sequence of a polypeptide that contains the partialregion consisting of Gly at position 205 to Phe at position 288 of theamino acid sequence shown in SEQ ID NO: 2, and consists of a fragment ofthe partial region consisting of the Ser at position 135 to Ile atposition 350 of the amino acid sequence shown in SEQ ID NO: 2, andencoding a polypeptide having activity of inducing dendritic-cell-likecell differentiation from monocytes;

(j) DNA encoding a polypeptide that consists of a partial region of theREIC/Dkk-3 protein and is capable of binding to the Tctex-1 protein,which consists of the nucleotide sequence consisting of g at position 4to g at position 69 of the nucleotide sequence shown in SEQ ID NO: 8;(k) DNA hybridizing under stringent conditions to DNA consisting of anucleotide sequence complementary to the nucleotide sequence consistingof g at position 4 to g at position 69 of the nucleotide sequence shownin SEQ ID NO: 8, and encoding a polypeptide having activity of bindingto the Tctex-1 protein; and(l) DNA encoding a polypeptide that consists of a partial region of theREIC/Dkk-3 protein, is capable of binding to the Tctex-1 protein,consists of the DNA sequence encoding the amino acid sequence shown inSEQ ID NO: 17, and has activity of binding to the Tctex-1 protein.[3] A vector, containing the DNA of [2].[4] The vector of [3], wherein the vector is an adenovirus vector.[5] An agent for inducing dendritic-cell-like cell differentiation frommonocytes, containing the polypeptide of [1] consisting of a partialregion of the REIC/Dkk-3 protein as an active ingredient.[6] An agent for accelerating the induction of differentiation toimmunoactivation cells selected from the group consisting of dendriticcells, helper T cells, CTL, and NK cells, containing the polypeptide of[1] consisting of a partial region of the REIC/Dkk-3 protein as anactive ingredient.[7] An agent for inhibiting the induction of differentiation toimmunosuppressively functioning cells that are myeloid-derivedsuppressor cells (MDSC) or immunoregulatory T cells (Treg), containingthe polypeptide of [1] consisting of a partial region of the REIC/Dkk-3protein as an active ingredient.[8] An anticancer agent, containing the polypeptide of [1] consisting ofa partial region of the REIC/Dkk-3 protein as an active ingredient.[9] An agent for inducing dendritic-cell-like cell differentiation frommonocytes, containing the DNA of [2] that encodes the polypeptideconsisting of a partial region of the REIC/Dkk-3 protein or the vectorof [3] or [4] as an active ingredient.[10] An agent for accelerating the induction of differentiation toimmunoactivation cells selected from the group consisting of dendriticcells, helper T cells, CTL, and NK cells, containing the DNA of [2]encoding the polypeptide consisting of a partial region of theREIC/Dkk-3 protein or the vector of [3] or [4] as an active ingredient.[11] An agent for inhibiting the induction of differentiation toimmunosuppressive cells that are myeloid-derived suppressor cells (MDSC)or immunoregulatory T cells (Treg), containing the DNA of [2] encodingthe polypeptide consisting of a partial region of the REIC/Dkk-3 proteinor the vector of [3] or [4] as an active ingredient.[12] An anticancer agent, containing the DNA of [2] encoding thepolypeptide consisting of a partial region of the REIC/Dkk-3 protein orthe vector of [3] or [4] as an active ingredient.[13] A method for inducing dendritic-cell-like cell differentiation fromCD14 positive monocytes, comprising culturing monocytes collected froman animal in vitro in the presence of the polypeptide of [1] consistingof a partial region of the REIC/Dkk-3 protein.[14] The method of [13], wherein the monocytes are peripheral bloodmonocytes.

This description includes part or all of the contents as disclosed inthe description and/or drawings of Japanese Patent Application Nos.2010-150935 and 2011-014319, which are priority documents of the presentapplication.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a method for preparing the full-length human REIC/Dkk-3protein and the partial regions 1 [Arg121-329] and 2 [Gly184-Ile329]thereof.

FIG. 2 shows photographs showing phase contrast microscopic images ofPBMCs cultured alone (no addition) (FIG. 2A), cultured in the presenceof GM-CSF+IL-4 (FIG. 2B) (2 ng/ml each), the full-length humanREIC/Dkk-3 protein (FIG. 2C), the partial region 1 [Arg 121-Ile 329](FIG. 2D), and the partial region 2 [Gly 184-Ile 329] (FIG. 2E) (10μg/ml) thereof, for 7 days (slightly expanded images), respectively.

FIG. 3 shows the phase contrast microscopic images of PBMCs culturedalone (no addition) (FIG. 3A), cultured in the presence of GM-CSF+IL-4(FIG. 3B) (2 ng/ml each), and the partial region 2 [Gly 184-Ile 329] (10μg/ml) (FIG. 3C) for 7 days (significantly expanded images),respectively.

FIG. 4 shows the frequency of the occurrence of dendritic-cell-likecells resulting from differentiation induction in the presence of cellsalone (no addition), GM-CSF+IL-4 (2 ng/ml each), the full-length humanREIC/Dkk-3 protein, the partial region 1 [Arg 121-Ile 329], or thepartial region 2 [Gly 184-Ile 329] (10 μg/ml).

FIG. 5-1 shows protocols for a production method and a purificationmethod for the partial region 3 [Ser 114-Phe 267] of a REIC/Dkk-3protein.

FIG. 5-2 shows a chart of purification by 1st ion exchangechromatography upon purification of the partial region 3 [Ser 114-Phe267] of the REIC/Dkk-3 protein.

FIG. 5-3 shows a chart of purification by 2^(nd) ion exchangechromatography upon purification of the partial region 3 [Ser 114-Phe267] of the REIC/Dkk-3 protein.

FIG. 6 shows photographs showing the induction of dendritic cell-likedifferentiation from peripheral blood monocytes through the addition ofa human REIC protein expressed by human tissue-derived cultured cells.Specifically, these photographs are slightly expanded phase-contrastmicroscopic images of dendritic-cell-like cells (on day 7) resultingfrom differentiation induction with the presence of cells alone (noaddition) (FIG. 6A), the presence of the partial region 1 [Arg 121-Ile329] (10 μg/ml) (FIG. 6B) or the partial region 3 [Ser 114-Phe 267] (10μg/ml) (FIG. 6C) of the REIC/Dkk-3 protein.

FIG. 7 shows photographs showing the induction of dendritic cell-likedifferentiation from peripheral blood monocytes through the addition ofthe human REIC protein expressed by human tissue-derived cultured cells.Specifically, these photographs are significantly expandedphase-contrast microscopic images of dendritic-cell-like cells (on day7) resulting from differentiation induction with the presence of cellsalone (no addition) (FIG. 7A), the partial region 1 [Arg 121-Ile 329](10μg/ml) (FIG. 7B) or the partial region 3 [Ser 114-Phe 267] (10 μg/ml)(FIG. 7C) of the REIC/Dkk-3 protein.

FIG. 8 shows the result of an experiment for confirming the stability ofthe partial region 3 [Ser 114-Phe 267] of a REIC/Dkk-3 protein.

FIG. 9 shows the construct of an expression cassette contained in ahigh-level gene expression plasmid used in Example 3.

FIG. 10 shows protocols for an experiment of the intraperitonealadministration of the human full-length REIC/Dkk-3 protein and thepartial region 3 [Ser 114-Phe 267].

FIG. 11A shows the effects of suppressing tumor growth (changes overtime in tumor volume) due to intraperitoneal administration of the humanfull-length REIC/Dkk-3 protein and the partial region 3 [Ser 114-Phe267].

FIG. 11B shows the effects of suppressing tumor growth (tumor weight)due to the intraperitoneal administration of the human full-lengthREIC/Dkk-3 protein and the partial region 3 [Ser 114-Phe 267].

FIG. 11C shows photographs showing tumors exhibiting the effects ofsuppressing tumor growth due to the intraperitoneal administration ofthe human full-length REIC/Dkk-3 protein and the partial region 3 [Ser114-Phe 267]. FIG. 11C-a shows the result of administration of PBS, FIG.11C-b shows the result of administration of the human full-lengthREIC/Dkk-3 protein, and FIG. 11C-c shows the result of administration ofthe partial region 3.

FIG. 12A is a graph showing the positive rate (%) of myeloid-derivedsuppressor cells in each type of peripheral blood at the time(immediately before euthanasia) of completion of an experiment oftreatment with a REIC/Dkk-3 protein (full-length or partial region 3),in an untreated group, or in a group treated with PBS buffer.

FIG. 12B is a graph showing the positive rate (%) of dendritic cells ineach type of peripheral blood at the time (immediately beforeeuthanasia) of completion of an experiment of treatment with theREIC/Dkk-3 protein (full-length or partial region 3), in an untreatedgroup, or in a group treated with PBS buffer.

FIG. 12C is a graph showing the positive rate (%) of activated dendriticcells (CD11c+/CD80+) in each type of peripheral blood at the time(immediately before euthanasia) of completion of an experiment oftreatment with the REIC/Dkk-3 protein (full-length or partial region 3),in an untreated group, or in a group treated with PBS buffer.

FIG. 12D is a graph showing the positive rate (%) of activated dendriticcells (CD11c+/CD86+) in each type of peripheral blood at the time(immediately before euthanasia) of completion of an experiment oftreatment with the REIC/Dkk-3 protein (full-length or partial region 3),in an untreated group, or in a group treated with PBS buffer.

FIG. 12E is a graph showing the positive rate (%) of helper T cells ineach type of peripheral blood at the time (immediately beforeeuthanasia) of completion of an experiment of treatment with theREIC/Dkk-3 protein (full-length or partial region 3), in an untreatedgroup, or in a group treated with PBS buffer.

FIG. 12F is a graph showing the positive rate (%) of immunosuppressive Tcells in each type of peripheral blood at the time (immediately beforeeuthanasia) of completion of an experiment of treatment with theREIC/Dkk-3 protein (full-length or partial region 3), in an untreatedgroup, or in a group treated with PBS buffer.

FIG. 12G is a graph showing the positive rate (%) of cytotoxic T cellsin each type of peripheral blood at the time (immediately beforeeuthanasia) of completion of an experiment of treatment with theREIC/Dkk-3 protein (full-length or partial region 3), in an untreatedgroup, or in a group treated with PBS buffer.

FIG. 12H is a graph showing the positive rate (%) of activated cytotoxicT cells in each type of peripheral blood at the time (immediately beforeeuthanasia) of completion of an experiment of treatment with theREIC/Dkk-3 protein (full-length or partial region 3), in an untreatedgroup, or in a group treated with PBS buffer.

FIG. 12I is a graph showing the positive rate (%) of NK cells in eachtype of peripheral blood at the time (immediately before euthanasia) ofcompletion of an experiment of treatment with the REIC/Dkk-3 protein(full-length or partial region 3), in an untreated group, or in a grouptreated with PBS buffer.

FIG. 13 shows protocols for an experiment of intraperitonealadministration of the human full-length REIC/Dkk-3 protein to orthotopicrenal cell carcinoma•pulmonary metastasis model mice.

FIG. 14A is a photograph showing the tumor tissue of primary lesions ofrenal cancer in an experiment of intraperitoneal administration of thehuman full-length REIC/Dkk-3 protein (10 μg, 100 μg) to orthotopic renalcell carcinoma•pulmonary metastasis model mice.

FIG. 14B is a graph showing the mean values of tumor weights (g) of theprimary lesions of renal cancer at the time of (immediately beforeeuthanasia) completion of an experiment of the treatment with thefull-length REIC/Dkk-3 protein (10 μg, 100 μg) or in a group treatedwith PBS buffer.

FIG. 15A shows photographs showing the tumor tissues of pulmonarymetastases in an experiment of intraperitoneal administration of thehuman full-length REIC/Dkk-3 protein (10 μg, 100 μg) to orthotopic renalcell carcinoma•pulmonary metastasis model mice.

FIG. 15B is a graph showing the mean values of tumor weights (g) ofpulmonary metastases at the time (immediately before euthanasia) ofcompletion of an experiment of treatment with the full-length REIC/Dkk-3protein (10 μg, 100 μg) or in a group treated with PBS buffer.

FIG. 16A shows cytograms showing the differentiation induction of MDSCwhen GM-CSF (20 ng/ml) (16A-a) or a mixture (16A-c) of a REIC protein(10 μg/ml) and GM-CSF (20 ng/ml) was administered to bone marrow cellscollected from untreated mice. 16A-a shows the result of a control.

FIG. 16B is a graph showing the positive rate (%) of MDSC found from thecytogram shown in FIG. 16A.

FIG. 17A-1 shows the result (cytogram) of measuring by flow cytometricanalysis the positive rate (%) of MDSC (Gr-1+, CD11b+) in each type ofperipheral blood collected at the time (immediately before euthanasia)of completion of an experiment of treatment with the full-lengthREIC/Dkk-3 protein (10 μg, 100 μg) or from a group treated with PBSbuffer. FIG. 17A-1 a shows the results of using PBS, FIG. 17A-1 b showsthe results of using the full-length REIC/Dkk-3 protein (10 μg), andFIG. 17A-1 c shows the results of using the full-length REIC/Dkk-3protein (100 μg).

FIG. 17A-2 shows the result (graph showing positive rate (%)) ofmeasuring by flow cytometric analysis the positive rate (%) of MDSC(Gr-1+, CD11b+) in each type of peripheral blood collected at the time(immediately before euthanasia) of completion of an experiment oftreatment with the full-length REIC/Dkk-3 protein (10 μg, 100 μg) orcollected from a group treated with PBS buffer.

FIG. 17B-1 shows the result (cytogram) of measuring by flow cytometricanalysis the positive rate of dendritic cells (CD11c+, CD80+) in eachtype of peripheral blood collected at the time (immediately beforeeuthanasia) of completion of an experiment of treatment with thefull-length REIC/Dkk-3 protein (10 μg, 100 μg) or collected from a grouptreated with PBS buffer. FIG. 17B-1 a shows the result of using PBS,FIG. 17B-1 b shows the result of using the full-length REIC/Dkk-3protein (10 μg), and FIG. 17B-1 c shows the result of using thefull-length REIC/Dkk-3 protein (100 μg).

FIG. 17B-2 shows the result (graph of positive rates) of measuring byflow cytometric analysis the positive rate of dendritic cells (CD11c+,CD80+) in each type of peripheral blood collected at the time(immediately before euthanasia) of completion of an experiment of thetreatment with full-length REIC/Dkk-3 protein (10 μg, 100 μg) orcollected from a group treated with PBS buffer.

FIG. 17C-1 shows the result (cytogram) of measuring by flow cytometricanalysis the positive rate of Treg (CD4+, Foxp3+) in each type ofperipheral blood collected at the time (immediately before euthanasia)of completion of an experiment of treatment with the full-lengthREIC/Dkk-3 protein (10 μg, 100 μg) or from a group treated with PBSbuffer. FIG. 17C-1 a shows the result of using PBS, FIG. 17C-1 b showsthe result of using the full-length REIC/Dkk-3 protein (10 μg), and FIG.17C-1 c shows the result of using the full-length REIC/Dkk-3 protein(100 μg).

FIG. 17C-2 shows the result (graph of positive rates) of measuring byflow cytometric analysis the positive rate of Treg (CD4+, Foxp3+) ineach type of peripheral blood collected at the time (immediately beforeeuthanasia) of completion of an experiment of treatment with thefull-length REIC/Dkk-3 protein (10 μg, 100 μg) or collected from a grouptreated with PBS buffer.

FIG. 17D-1 shows the result (cytogram) of measuring by flow cytometricanalysis the positive rate of activated CTL (CD8+, CD69) in each type ofperipheral blood collected at the time (immediately before euthanasia)of completion of an experiment of treatment with the full-lengthREIC/Dkk-3 protein (10 μg, 100 μg) or collected from a group treatedwith PBS buffer. FIG. 17D-1 a shows the result of using PBS, FIG. 17D-1b shows the result of using the full-length REIC/Dkk-3 protein (10 μg),and FIG. 17D-1 c shows the result of using the full-length REIC/Dkk-3protein (100 μg).

FIG. 17D-2 shows the result (graph of positive rates) of measuring byflow cytometric analysis the positive rate of activated CTL (CD8+,CD69+) in each type of peripheral blood collected at the time(immediately before euthanasia) of completion of an experiment oftreatment with the full-length REIC/Dkk-3 protein (10 μg, 100 μg) orcollected from a group treated with PBS buffer.

FIG. 17E-1 shows the result (cytogram) of measuring by flow cytometricanalysis the positive rate of NK cells (CD3e+, NK1.1+) in each type ofperipheral blood collected at the time (immediately before euthanasia)of completion of an experiment of treatment with the full-lengthREIC/Dkk-3 protein (10 μg, 100 μg) or collected from a group treatedwith PBS buffer. FIG. 17E-1 a shows the result of using PBS, FIG. 17E-1b shows the result of using the full-length REIC/Dkk-3 protein (10 μg),and FIG. 17E-1 c shows the result of using the full-length REIC/Dkk-3protein (100 μg).

FIG. 17E-2 shows the result (graph of positive rates) of measuring byflow cytometric analysis the positive rate of NK cells (CD3e+, NK1.1+)in each type of peripheral blood collected at the time (immediatelybefore euthanasia) of completion of an experiment of treatment with thefull-length REIC/Dkk-3 protein (10 μg, 100 μg) or collected from a grouptreated with PBS buffer.

FIG. 18A shows the result of yeast 2-hybrid analysis, exhibiting aninteraction between a REIC/Dkk-3 protein and a Tctex-1 protein. In FIG.18A, blue colonies indicate the presence of the interaction between theREIC/Dkk-3 protein and the Tctex-1 protein.

FIG. 18B shows the results of immunoprecipitation analysis, exhibitingan interaction between the REIC/Dkk-3 protein and the Tctex-1 proteinand the results of Western blot analysis.

FIG. 19A shows the results of animal cell 2-hybrid analysis, exhibitingan interaction between the full-length REIC/Dkk-3 protein and theTctex-1 protein.

FIG. 19B shows the results of animal cell 2-hybrid analysis, exhibitingan interaction between a partial region of the human full-lengthREIC/Dkk-3 protein and the Tctex-1 protein.

FIG. 20A shows the amino acid sequence alignment of a REIC/Dkk-3 proteinwith a TcTex-1 binding region of a dynein intermediate chain (DIC).

FIG. 20B shows the amino acid sequence alignment of the Tctex bindingdomain of the REIC/Dkk-3 protein with a known binding protein.

FIG. 21A shows photographs showing intracellular localization of thehuman full-length REIC/Dkk-3 protein and the Tctex-1 protein.Photographs in FIG. 21A were taken with a confocal microscope afterdouble fluorescent staining (A-c) of the human full-length REIC/Dkk-3protein (A-b) with concanavalin A (A-a) as an endoplasmic reticulummarker in human OUMS24 fibroblasts.

FIG. 21B shows photographs showing intracellular localization of thehuman full-length REIC/Dkk-3 protein and the Tctex-1 protein.Photographs in FIG. 21B were taken with a confocal microscope afterdouble fluorescent staining (B-c) of the Tctex-1 protein (B-b) withconcanavalin A (B-a) as an endoplasmic reticulum marker in human OUMS24fibroblasts.

FIG. 22A shows photographs showing images taken by fluorescencemicroscopy after Hochest staining, which indicate that the capacity ofAd-REIC to induce apoptosis was decreased by a decreased Tctex-1expression level, but was enhanced by amplified Tctex-1 expression.Photographs on the top show the results of using Ad-LacZ, photographs onthe bottom show the results of using Ad-REIC, and “a,” “b,” and “c”indicate the results of administration of a GFP plasmid, shRNA-Tctex-1,and a Tctex-1 expression plasmid, respectively.

FIG. 22B is a graph showing apoptosis induction rates, indicating thatthe capacity of Ad-REIC to induce apoptosis was decreased by a decreasedTctex-1 expression level, but was enhanced by amplified Tctex-1expression.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Hereafter, the present invention is described in detail.

The full-length nucleotide sequence of a REIC/Dkk-3 gene (REIC gene) andthe amino acid sequence of the protein encoded by the gene are shown inSEQ ID NO: 1 and SEQ ID NO: 2, respectively. In the amino acid sequenceshown in SEQ ID NO: 2, the sequence consisting of amino acids 1 to 21 ispredicted to be a signal sequence. The REIC/Dkk-3 gene can be obtainedfrom human cells, human tissues, and the like based on the sequenceinformation of SEQ ID NO: 1. The REIC/Dkk-3 gene can also be obtainedaccording to the International Publication WO01/038523 pamphlet.

Examples of the polypeptide consisting of a partial region of theREIC/Dkk-3 protein (REIC protein) of the present invention include thefollowing polypeptides.

(1) A polypeptide consists of 209 amino acids containing the partialregion consisting of Arg at position 121 to Ile at position 329 preparedby removing a signal sequence portion from the REIC/Dkk-3 protein. Thepolypeptide consists of Arg at position 142 to Ile at position 350 ofthe amino acid sequence shown in SEQ ID NO: 2. The polypeptide of thepresent invention may also be referred to as the partial region [Arg121-Ile 329 (Arg at position 121-Ile at position 329] of the REIC/Dkk-3protein. The amino acid sequence of the partial region [Arg 121-Ile 329]of the REIC/Dkk-3 protein of the present invention is shown in SEQ IDNO: 5 and the nucleotide sequence encoding the amino acid sequence isshown in SEQ ID NO: 6.(2) A polypeptide consists of 146 amino acids containing the partialregion consisting of Gly at position 184 to Ile at position 329 preparedby removing a signal sequence portion from the REIC/Dkk-3 protein. Thepolypeptide consists of Gly at position 205 to Ile at position 350 ofthe amino acid sequence shown in SEQ ID NO: 2. The polypeptide of thepresent invention may also be referred to as the partial region [Gly184-Ile 329] of the REIC/Dkk-3 protein. The amino acid sequence of thepartial region [Gly 184-Ile 329] of the REIC/Dkk-3 protein of thepresent invention is shown in SEQ ID NO: 3 and the nucleotide sequenceencoding the amino acid sequence is shown in SEQ ID NO: 4.(3) A polypeptide consists of 154 amino acids containing the partialregion consisting of Ser at position 114 to Phe at position 267 preparedby removing a signal sequence portion from the REIC/Dkk-3 protein. Thepolypeptide consists of Ser at position 135 to Phe at position 288 ofthe amino acid sequence shown in SEQ ID NO: 2. The polypeptide of thepresent invention may also be referred to as the partial region [Ser114-Phe 267] of the REIC/Dkk-3 protein. The amino acid sequence of thepartial region [Ser 114-Phe 267] of the REIC/Dkk-3 protein of thepresent invention is shown in SEQ ID NO: 7 and the nucleotide sequenceencoding the amino acid sequence is shown in SEQ ID NO: 8.(4) The polypeptide consists of 83 amino acids containing the partialregion consisting of Gly at position 184 to Phe at position 267 preparedby removing a signal sequence portion from the REIC/Dkk-3 protein. Thepolypeptide consists of the consensus sequence of the 3 above types ofpolypeptide, and is thought to play a core part of bioactivity. Thepolypeptide consists of Gly at position 205 to Phe at position 288 ofthe amino acid sequence shown in SEQ ID NO: 2. The polypeptide of thepresent invention may also be referred to as the partial region [Gly184-Phe 267] of the REIC/Dkk-3 protein. The amino acid sequence of thepartial region [Gly 184-Phe 267] of the REIC/Dkk-3 protein of thepresent invention is shown in SEQ ID NO: 9 and the nucleotide sequenceencoding the amino acid sequence is shown in SEQ ID NO: 10.(5) Another example of the polypeptide of the present inventionconsisting of a partial region of the REIC/Dkk-3 protein (REIC protein)is a polypeptide containing the partial region consisting of Gly atposition 184 to Phe at position 267 prepared by removing a signalsequence portion from the REIC/Dkk-3 protein, and consists of a fragmentof the partial region consisting of Ser at position 114 to Ile atposition 329. The polypeptide contains the partial region consisting ofGly at position 205 to Phe at position 288 of the amino acid sequenceshown in SEQ ID NO: 2, and consists of a fragment of the partial regionconsisting of Ser at position 135 to Ile at position 350 of the aminoacid sequence shown in SEQ ID NO: 2. Also, the nucleotide sequenceencoding the polypeptide contains the partial sequence consisting of gat position 613 to c at position 864 of the nucleotide sequence shown inSEQ ID NO: 1, and consists of a fragment of the partial sequenceconsisting of t at position 403 to t at position 1050 of the nucleotidesequence shown in SEQ ID NO: 1. The number of amino acid residues of thepolypeptide ranges from 83 to 216.

Furthermore, the REIC/Dkk-3 protein interacts (association) with aTctex-1 (t-complex testis expressed-1) protein to act. The Tctex-1protein is a light chain protein (dynein light chain) composing a dyneinmotor complex, and plays an important role as an intervening moleculebetween the dynein motor and vesicular load through association with aTctex-1 binding protein.

Both REIC/Dkk-3 protein and Tctex-1 protein are localized around theendoplasmic reticulum. The Tctex-1 protein accelerates the capacity ofthe REIC/Dkk-3 protein to induce apoptosis. The partial region of theREIC/Dkk-3 protein consists of 22 amino acids consisting of Val atposition 136 to Met at position 157 of the amino acid sequence shown inSEQ ID NO: 2 and interacts with the Tctex-1 protein. The partial regionpolypeptide corresponds to the partial region consisting of Val atposition 115 to Met at position 136 prepared by removing the signalsequence portion from the REIC/Dkk-3 protein. The amino acid sequence ofthe partial region is shown in SEQ ID NO: 17.

In the amino acid sequence of the partial region, EXGRRXH (correspondsto the sequence consisting of amino acids 4 to 10 of SEQ ID NO: 18 andSEQ ID NO: 17) (X denotes an arbitrary natural amino acid) is aconsensus sequence (consensus motif) involved in binding with theTctex-1 protein.

The present invention encompasses the partial region peptide of theREIC/Dkk-3 protein interacting with the above Tctex-1 protein and theabove consensus motif.

Therefore, an example of the polypeptide of the present inventionconsisting of a partial region of the REIC/Dkk-3 protein (REIC protein)is a polypeptide consisting of a partial region of the REIC/Dkk-3protein, being capable of binding to the Tctex-1 protein, and consistingof the amino acid sequence shown in SEQ ID NO: 17. Another examplethereof is a polypeptide consisting of 7 to 22 amino acid residuescontaining the sequence of the consensus motif shown in the aboveEXGRRXH (SEQ ID NO: 18). The polypeptide consists of continuous 7 to 22amino acid residues in the amino acid sequence shown in SEQ ID NO: 17and contains the amino acid sequence consisting of 7 amino acid residuesof Glu at position 4 to H is at position 10. In this case, Glu atposition 5 and Ser at position 9 may be substituted with any othernatural amino acids.

The partial region polypeptide of the REIC/Dkk-3 protein of the presentinvention contains the above amino acid sequence; that is, the aminoacid sequence shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQID NO: 9, or the partial region consisting of Gly at position 184 to Pheat position 267 prepared by removing a signal sequence portion from theREIC/Dkk-3 protein, has the amino acid sequence of a polypeptideconsisting of a fragment of the partial region consisting of Ser atposition 114 to Ile at position 329 or an amino acid sequencesubstantially the same as the amino acid sequence, and has activity ofinducing dendritic-cell-like cell differentiation. Also, the partialregion polypeptide of the REIC/Dkk-3 protein of the present inventionhas the amino acid sequence shown in SEQ ID NO: 17 or an amino acidsequence substantially the same as the amino acid sequence, and hasactivity of binding to the Tctex-1 protein. Here, examples of such anamino acid sequence substantially the same as the above-mentioned aminoacid sequence include: an amino acid sequence having a substitution, adeletion and/or an addition of 1 or a plurality of or several (1 to 10,preferably 1 to 5, and further preferably 1 or 2) amino acids withrespect to the amino acid sequence, and an amino acid sequence having atleast 85% or more, preferably 90% or more, further preferably 95% ormore, and particularly preferably 97% or more identity with the aminoacid sequence when calculated using BLAST (Basic Local Alignment SearchTool at the National Center for Biological Information (the basic localalignment search tool (BLAST) of the National Center for BiotechnologyInformation (NCBI))) or the like (e.g., using default; that is,initially set parameters).

The polypeptide of the present invention has activity of inducingdendritic-cell-like cell differentiation from monocytes, has activity ofinducing the differentiation to immunoactivation cells such as CTLcells, NK cells, and helper T cells, and further has activity ofsuppressing MDSC and Treg cell differentiation. The polypeptide of thepresent invention has activity of inducing or suppressingdifferentiation to these cells, and thus it is able to inhibit theimmunosuppression system. Therefore, the polypeptide of the presentinvention can be used as an anticancer immunostimulator, an anticanceragent, an antitumor agent, an agent for inducing or suppressing immunecell differentiation, or the like.

DNA encoding the partial region polypeptide of the REIC/Dkk-3 protein ofthe present invention encodes a protein having activity of: inducingdendritic-cell-like cell differentiation; inducing the differentiationto immunoactivation cells such as CTL cells, NK cells, and helper Tcells; or suppressing MDSC and Treg cell differentiation, and is: DNAcontaining the nucleotide sequence shown in SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, or SEQ ID NO: 10, the nucleotide sequence consisting of gat position 4 to g at position 69 of the nucleotide sequence shown inSEQ ID NO: 8, or the partial sequence consisting of g at position 613 toc at position 864 of the nucleotide sequence shown in SEQ ID NO: 1, andhybridizing under stringent conditions to DNA having a nucleotidesequence complementary to the nucleotide sequence consisting of afragment of the partial sequence consisting of t at position 403 to t atposition 1050 of the nucleotide sequence shown in SEQ ID NO: 1;

DNA containing the nucleotide sequence consisting of g at position 4 tog at position 69 of the nucleotide sequence shown in SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or the partial sequenceconsisting of g at position 613 to c at position 864 of the nucleotidesequence shown in SEQ ID NO: 1, and having at least 85%, preferably 90%or more, further preferably 95% or more, and particularly preferably 97%or more identity with the nucleotide sequence consisting of a fragmentof the partial sequence consisting of t at position 403 to t at position1050 of the nucleotide sequence shown in SEQ ID NO: 1 when calculatedusing BLAST (Basic Local Alignment Search Tool at the National Centerfor Biological Information (the basic local alignment search tool(BLAST) of the National Center for Biotechnology Information (NCBI))) orthe like (e.g., using default; that is, initially set parameters); orDNA encoding a protein consisting of an amino acid sequence having asubstitution, a deletion and/or an addition of 1 or a plurality of orseveral (1 to 10, preferably 1 to 5, and further preferably 1 or 2)amino acids with respect to the amino acid sequence of the proteinencoded by the above DNA. Here, the term “stringent conditions” refersto conditions of about “1×SSC, 0.1% SDS, and 37° C.,” more stringentconditions refers to conditions of about “0.5×SSC, 0.1% SDS, and 42°C.,” and further stringent conditions refers to conditions of about“0.2×SSC, 0.1% SDS, and 65° C.”

A partial region polypeptide of the REIC/Dkk-3 protein can be obtainedby chemical synthesis based on the above sequence information. Also, apartial region polypeptide of the REIC/Dkk-3 protein can be obtained asa recombinant polypeptide by a genetic engineering technique.Specifically, DNA encoding a partial region polypeptide of theREIC/Dkk-3 protein of the present invention is introduced into anappropriate vector, the vector is inserted into a host, the host iscultured, and then the polypeptide can be obtained from the cultureproduct. Examples of a vector to be used for insertion of the DNA of thepresent invention are not particularly limited as long as it isreplicable within a host and include plasmid DNA and phage DNA. Knownvectors can be used herein. At this time, as hosts, eukaryotic celllines or prokaryotic cell lines can be used. Examples of eukaryoticcells include animal cells such as established mammalian (e.g., human orrodent) cell lines, insect cell lines, fungal cells (e.g., filamentouscells) and yeast cells. Examples of prokaryotic cells include bacterialcells such as Escherichia coli cells. Host cells containing DNA encodingthe partial region polypeptide of the REIC/Dkk-3 protein of the presentinvention are cultured in vitro or in vivo; that is, the host iscultured by a known method, so that a partial region polypeptide of theREIC/Dkk-3 protein can be obtained from the culture product. Here, theterm “culture product” refers to any of a culture supernatant, culturedcells, cultured microorganisms, disrupted cells, and disruptedmicroorganisms. The thus expressed and produced polypeptide can bepurified from the culture product. Purification may be performed by ageneral purification method employed for proteins. For example,purification can be performed by appropriately selecting and combiningion exchange chromatography, affinity chromatography, gel filtration,ultrafiltration, salting-out, dialysis, and the like. Moreover, thepartial region polypeptide of the REIC/Dkk-3 protein of the presentinvention can also be obtained according to WO01/038523.

Among examples of the partial region polypeptide of the REIC/Dkk-3protein of the present invention, particularly the partial region [Gly184-Phe 267] of the REIC/Dkk-3 protein is stable such that it is notdegraded even when stored at room temperature to an about hightemperature of 37° C. The partial region polypeptide is also stableagainst various reagents such as PEG.

The present invention further encompasses a vector containing the aboveDNA encoding the partial region polypeptide of the REIC/Dkk-3 protein.The vector is introduced into a subject, the partial region polypeptideof the REIC/Dkk-3 protein is expressed in vivo within the subject, andthus the partial region polypeptide can exhibit bioactivity in vivo.

The partial region polypeptide of the REIC/Dkk-3 protein inducesapoptosis in cancer cells.

In genetic therapy, the target gene (DNA) can be introduced into thesubject in accordance with a known technique. Examples of techniques forintroducing a gene into a subject include a method involving the use ofa virus vector and a method involving the use of a non-virus vector.Various techniques are known (Bessatsu Jikken-Igaku,Idenshi-Chiryo-No-Kisogijutsu (Basic Techniques for Gene Therapy),Yodosha Co., Ltd., 1996; Bessatsu Jikken Igaku (Separate volume,Experimental Medicine), Idenshi donyu & hatsugen kaiseki jikken-hou(Experimentation of gene introduction & expression analysis), Yodosha,Co., Ltd. 1997; and the Japan Society of Gene Therapy (ed.), “Idenshichiryo kaihatsu kenkyu handbook (the Handbook for research anddevelopment of gene therapy),” N.T.S., 1999).

Representative examples of virus vectors used for gene introductioninclude an adenovirus vector, an adeno-associated virus vector, and aretrovirus vector. A target gene may be introduced into a cell byintroducing a target gene into a DNA or RNA virus, such as a detoxicatedretrovirus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbisvirus, Sendai virus, SV40, or HIV, and infecting the cell with suchvirus.

When the gene according to the present invention is used for genetictherapy using a virus, an adenovirus vector is preferably used. Anadenovirus vector is characterized in that: (1) it can introduce genesinto many types of cells; (2) it can efficiently introduce genes intocells at the period of growth arrest; (3) it enables concentration viacentrifugation to yield high-titer viruses (10 to 11 PFU/ml or more);and (4) it is suitable for direct gene introduction into tissue cells invivo. As adenovirus vectors used for genetic therapy, a first-generationadenovirus vector lacking the E1/E3 region (Miyake, S. et al., Proc.Natl. Acad. Sci., U.S.A., 93, 1320, 1996), the second-generationadenovirus vector prepared from the first-generation adenovirus vectorby deleting the E2 or E4 region in addition to the E1/E3 region (Lieber,A. et al., J. Virol., 70, 8944, 1996; Mizuguchi, H. & Kay, M. A., Hum.Gene Ther., 10, 2013, 1999), and the third-generation adenovirus vectorlacking substantially all the adenovirus genome (GUTLESS) (Steinwaerder,D. S., et al., J. Virol., 73, 9303, 1999) have been developed. The geneaccording to the present invention can be introduced with the use of anyof such adenovirus vectors without particular limitation. Further, theadeno-AAV hybrid vector to which the capacity for incorporating the geneinto the AAV chromosome has been imparted (Recchia, A. et al., Proc.Natl. Acad. Sci., U.S.A., 96, 2615, 1999) or an adenovirus vectorcapable of incorporating the gene into the chromosome with the use of atransposon gene may be used, so that such vector can be applied tolong-term gene expression. Also, a peptide sequence exhibitingtissue-specific transferability to the H1 loop of the adenovirus fibermay be inserted to impart tissue specificity to the adenovirus vector(Mizuguchi, H. & Hayakawa, T., Nippon Rinsho, 7, 1544, 2000).

Alternatively, the target gene can be introduced into a cell or tissueusing a recombinant expression vector into which a gene expressionvector, such as a plasmid vector, has been incorporated, without the useof the above viruses. For example, a gene can be introduced into a cellvia lipofection, calcium phosphate coprecipitation, a DEAE-dextranmethod, or direct injection of DNA using a micro glass tube. Also, arecombinant expression vector can be incorporated into a cell via, forexample, gene introduction using an internal liposome, gene introductionusing an electorostatic type liposome, a method using HVJ-liposome, amethod using a modified HVJ-liposome (i.e., the HVJ-AVE liposomemethod), a method using an HVJ-E (envelope) vector, receptor-mediatedgene introduction, a method in which a particle gun is used to introduceDNA molecules in a cell with a carrier (i.e., a metal particle), directintroduction of naked-DNA, or gene introduction using various types ofpolymers. In such a case, any expression vector can be used, providedthat such vector can express the target gene in vivo. Examples of suchvectors include pCAGGS (Gene 108, 193-200, 1991), pBK-CMV, pcDNA3, 1,and pZeoSV (Invitrogen, Stratagene), and pVAX1 vectors.

A vector comprising DNA encoding a partial region polypeptide of theREIC/Dkk-3 protein may adequately comprise a promoter or enhancer fortranscribing the gene, poly A signal, a marker gene for labeling and/orselecting the cell into which the gene has been introduced, and thelike. In such a case, a known promoter can be used.

A gene therapeutic agent containing DNA encoding a partial regionpolypeptide of the REIC/Dkk-3 protein of the present invention may beintroduced into a subject by, for example, the in vivo method wherein agene therapeutic agent is directly introduced into a body or the ex vivomethod wherein a given cell is extracted from a human, a genetherapeutic agent is introduced into the cell ex vivo, and the cell isthen returned into the body (Nikkei Science, April 1994, pp. 20-45;Gekkan Yakuji (Japan Medicine Monthly), 36(1), 23-48, 1994; Jikken igakuzoukan (special issue, Experimental Medicine), 12(15), 1994; the JapanSociety of Gene Therapy (ed.), Idenshi chiryo kaihatsu kenkyu (Studiesfor Development of Gene Therapy) handbook, N. T. S., 1999).

The partial region polypeptide of the REIC/Dkk-3 protein, DNA encodingthe polypeptide, and the vector containing the DNA of the presentinvention can be used as remedies or reagents.

The partial region polypeptide of the REIC/Dkk-3 protein of the presentinvention, DNA encoding the polypeptide, and a vector containing the DNAcan induce dendritic-cell-like cell differentiation from monocytes, canenhance anticancer immune activity, and can be used for cancertreatment.

The partial region polypeptide of the REIC/Dkk-3 protein of the presentinvention, DNA encoding the polypeptide, and a vector containing the DNAcan be used as agents for inducing dendritic-cell-like celldifferentiation from monocytes, agents for activating cancer immunity,and pharmaceutical compositions having the effect of activating cancerimmunity for cancer treatment or cancer prevention. Here, the term“activity of inducing dendritic-cell-like cell differentiation frommonocytes” refers to activity of acting on monocytes to differentiatethem into dendritic-cell-like cells. Whether or not dendritic-cell-likecell differentiation is induced by the addition of the partial regionpolypeptide of the REIC/Dkk-3 protein can be detected based onmorphological features and surface antigens. Specifically, such featuresof the dendritic-cell-like cells are: the cells have morphologicallydendrites; and the cells are found to be positive for dendritic cellmarkers, CD11c, CD40, CD80, CD86, and HLA-DR as surface antigens, asanalyzed by flow cytometry.

Furthermore, a vector containing DNA encoding the partial regionpolypeptide of the REIC/Dkk-3 protein is introduced into a subject, thepartial region polypeptide of the REIC/Dkk-3 protein is expressed invivo within the subject, and thus the partial region polypeptide canexhibit an effect of inducing dendritic-cell-like cell differentiationfrom monocytes and an effect of treating or preventing cancer by theeffect of activating cancer immunity and the action of activating cancerimmunity.

Monocytes used in the present invention include peripheral blood-derivedmonocytes, bone marrow-derived monocytes, splenocyte-derived monocytes,and umbilical cord blood-derived monocytes. Of these, peripheralblood-derived monocytes are preferable. When specific monocytes such asCD14 positive monocytes are collected from a living body and induced bythe partial region polypeptide of the REIC/Dkk-3 protein todifferentiate into dendritic cell-like cells, such monocytes can becollected by an FACS (fluorescent activated cell sorter), a flowcytometer, or the like with the use of the presence of CD14 as an index.The animal species that is the origin of monocytes is not limited.Examples of animals that can be used include mammals such as mice, rats,guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, bovines,horses, goats, monkeys, and humans. Isolation of a specific cellpopulation by an FACS can be carried out by a known method. As an FACSor a flow cytometer, an FACS vantage (Becton, Dickinson and Company), anFACS Calibur (Becton, Dickinson and Company), or the like can be used,for example.

Monocytes can be cultured by a known technique for culturing humanlymphoid cells. For a culture solution, for example, a known base mediumsuch as RPMI1640 or DMEM can be used. Culture may be carried out byadding an appropriate antibiotic, animal serum, or the like to such basemedium. Culture vessels used herein are not limited. Commerciallyavailable plates, dishes, and flasks can be adequately selected and useddepending on the culture scale.

The present invention includes a method for culturing monocytes in vitroin the presence of the REIC protein and inducing dendritic cell-likecell differentiation from monocytes. In the method of the presentinvention, for example, culture may be carried out using monocytes at aconcentration of 10⁴ to 10⁷ cells/ml with the addition of the partialregion polypeptide of the REIC/Dkk-3 protein at a concentration of 1 to20 μg/ml.

Dendritic cells play a very important role in the mechanisms of cancerimmunity, inflammation, and the like in vivo. Dendritic cell-like cellsinduced to differentiate by the REIC/Dkk-3 protein according to themethod of the present invention are morphologically similar to dendriticcells induced by IL-4+GM-CSF. However, to be exact, the dendriticcell-like cells differ from such dendritic cells, and therefore they arenovel dendritic cell-like cells. Dendritic cell-like cells induced bythe partial region polypeptide of the REIC/Dkk-3 protein are indendritic forms. In addition, the dendritic cell-like cells are positivefor dendritic cell markers such as CD 11c, CD40, CD80, CD86, and HLA-DR.In this regard, novel dendritic cell-like cells of the present inventioncan be classified as dendritic cells. However, they are negative forCD1a, which is a dendritic cell marker, and positive for CD14, for whichdendritic cells are generally supposed to be negative.

In the case of induction from CD14 positive monocytes with stimulationwith the partial region polypeptide of the REIC/Dkk-3 protein, it refersto “dendritic cell-like differentiated cells that have been activated bythe REIC protein (REIC activated monocytes with dendritic cellfeatures).”

The partial region polypeptide of the REIC/Dkk-3 protein of the presentinvention has the capacity of inducing dendritic-cell-like celldifferentiation higher than that of the full-length REIC/Dkk-3 protein.

The present invention encompasses dendritic cell-like cells induced fromCD14 positive monocytes by the partial region polypeptide of theREIC/Dkk-3 protein.

Dendritic cell-like cells obtained via induction by the partial regionpolypeptide of the REIC/Dkk-3 protein can be used for cancerimmunotherapy. Specifically, monocytes are collected from a subject, themonocytes are cultured with the partial region polypeptide of theREIC/Dkk-3 protein, the dendritic cell-like cells are induced, and thenthe obtained dendritic cell-like cells are returned to the subject.Thus, dendritic cell-like cells themselves can be used for cancertreatment or prevention, or the like. In such case, dendritic cell-likecells induced by the partial region polypeptide of the REIC/Dkk-3protein act in a non-cancer-type-specific manner and exhibit cancerimmunotherapeutic effects. However, it is also possible to add acancer-type specific tumor antigen or an autologous tumor lysate uponinduction of dendritic cell-like cells. In addition, induced dendriticcell-like cells can be cocultured with a specific tumor antigen or anautologous tumor lysate. It becomes possible to attack cancer cells in atumor-specific manner by stimulating dendritic cell-like cells with acancer-type-specific tumor antigen or an autologous tumor lysate.

Dendritic cell-like cells can be intradermally, subcutaneously,intravenously, or intralymphaticaly administered.

In addition, the partial region polypeptide of the REIC/Dkk-3 protein isthought to have a cytokine-like activity such that it acts on cells inan extracellular manner so as to control cell differentiation.Therefore, it is believed that the partial region polypeptide of theREIC/Dkk-3 protein widely functions in vivo in relation to immunity andinflammation. Thus, the partial region polypeptide of the REIC/Dkk-3protein or DNA encoding the same can be administered to a subject as anagent for inducing differentiation into dendritic cell-like cells or anagent for activating dendritic cell-like cells for in vivo use. Thepartial region polypeptide of the REIC/Dkk-3 protein induces dendriticcell-like cells in a subject. As a result, the dendritic cell-like cellssystematically activate lymphocytes having anticancer activity in thesubject, resulting in the exhibition of cancer immune effects.Therefore, the partial region polypeptide of the REIC/Dkk-3 protein orDNA encoding the same can be used as an agent for activating cancerimmunity. Further, since the partial region polypeptide of theREIC/Dkk-3 protein-induced dendritic cell-like cells have cancer immuneeffects, the partial region polypeptide of the REIC/Dkk-3 protein or DNAencoding the same can be used as a pharmaceutical composition for cancertreatment or prevention (a cancer immunotherapeutic agent). In suchcase, the partial region polypeptide of the REIC/Dkk-3 protein or DNAencoding the same may be administered alone. In this case, the effectsare exhibited in a non-cancer-type-specific manner. Alternatively, itmay be administered with a specific tumor antigen. In such a case, theeffects can be exhibited in a cancer-type-specific manner.

Also, the REIC/Dkk-3 protein concentration in tissue in whichcanceration progresses is low, anticancer immune activity is unlikely tobe induced in cancer tissue, so that the presence of cancer is notdetected through the biological immunity (cancer cell immunologicaltolerance), resulting in proliferation or worsening of cancer. Undersuch circumstances, the partial region polypeptide of the REIC/Dkk-3protein of the present invention is not only useful as a cancertherapeutic agent (an anticancer agent or an antitumor agent), but alsouseful as an agent for preventing canceration/carcinogenesis throughanticancer immune activation. The partial region polypeptide is alsouseful as a cancer immunotherapeutic agent.

Furthermore, the partial region polypeptide of the REIC/Dkk-3 protein,DNA encoding the polypeptide, and a vector containing the DNA haveactivity of inducing the differentiation to immunoactivation cells suchas CTL (cytotoxic T lymphocyte), NK (Natural killer) cells, and helper Tcells, and activity of suppressing the differentiation to MDSC (myeloidderived suppressor cells: myelocyte-derived suppressor cells) and Tregcells (regulatory T cells). The polypeptide of the present invention canenhance anticancer immune activity, can be used for cancer treatment,and can exhibit anticancer effects on local and metastatic foci ofcancer. Specifically, the polypeptide of the present invention can beused as an anticancer agent, an antitumor agent, an agent for activatinganticancer immunity, an agent for potentiating systemic immunity or anagent for inducing the differentiation to immunoactivation cells, whichinvolves accelerated induction of the differentiation toimmunoactivation cells represented by dendritic cells, helper T cells,CTL, and NK cells, and as an agent for inhibiting the induction ofdifferentiation to immunosuppressive cells represented by MDSC and Tregcells.

Examples of a cancer that is treated or prevented using the remedy ofthe present invention include cranial nerve tumor, skin cancer, gastriccancer, lung cancer, hepatic cancer, lymphoma/leukemia, colon cancer,pancreatic cancer, anal/rectal cancer, esophageal cancer, uterinecancer, breast cancer, adrenal cancer, kidney cancer, renal pelvic andureteral cancer, bladder cancer, prostate cancer, urethral cancer,penile cancer, testicular cancer, osteoma/osteosarcoma, leiomyoma,rhabdomyoma, and mesoepithelioma. In particular, breast cancer andbladder cancer are preferable.

The remedy of the present invention contains DNA encoding the partialregion polypeptide of the REIC/Dkk-3 protein, a vector containing theDNA, or the polypeptide encoded by the DNA, and a pharmacologicallyacceptable carrier, diluent, or excipient. The remedy of the presentinvention for cancer treatment or prevention can be administered in avariety of dosage forms. Examples of dosage forms include: tablets,capsules, granules, powders, and syrups for peroral administration; andinjection preparations (e.g., for subcutaneous injection, intravenousinjection, intramuscular injection, and intraperitoneal injection),drops, suppositories, spray, eye drops, transnasal preparations,transdermal preparations, transmucosal preparations, transpulmonarypreparations, and adhesive preparations for parenteral administration.

The remedy of the present invention can also be used for systemicmedication through injection or the like, or, can also be used for localadministration. For example, the remedy is administered to a cancer sitevia injection, so as to be able to exhibit its effects. In particular,in the case of local administration of a vector containing DNA encodingthe partial region polypeptide of the REIC/Dkk-3 protein, the peptide isproduced by the vector for a long period of time at the cancer site, soas to be able to exhibit the effects.

Preferably, the remedy is directly injected locally to a cancer lesiononce or multiple times so that the agent reaches all parts of the cancerlesion.

The remedy of the present invention comprises a carrier, a diluent, andan excipient that are generally used in the drug manufacturing field.For example, lactose or magnesium stearate can be used as a carrier orexcipient of a tablet. Physiological saline or an isotonic solutioncontaining glucose and another adjuvant is used as an aqueous solutionof an injection, for example. Such an aqueous solution may be used incombination with an adequate solubilizer, such as alcohol, a polyalcoholsuch as propylene glycol, or a nonionic surfactant. Sesame oil, soybeanoil, or the like is used as an oily liquid, and, as a solubilizer,benzyl benzoate, benzyl alcohol, or the like may be used in combination.

The dose varies depending on symptoms, age, body weight, and otherconditions. In the case of the remedy, a dose may be 0.001 mg to 100 mgthereof at intervals of several days, several weeks, or several months,and it may be administered via subcutaneous injection, intramuscularinjection, or intravenous injection. In the case of using a vectorcontaining DNA encoding the partial region polypeptide of the REIC/Dkk-3protein, 10⁷ to 10⁹ pfu (plaque forming unit) of the vector may beadministered, for example.

The remedy of the present invention is also effective for a cancerpatient having a cancer lesion confirmed to exhibit resistance tovarious existing treatments such as treatment with an anticancer agent.

The remedy of the present invention is confirmed to exhibit the effectsof cancer cell death and/or tumor shrinkage even through single-agentadministration thereof. Moreover, the combined use of this agent with ananticancer agent doubly induces anticancer effects, so that a strongtumor shrinkage effect can be expected.

Furthermore, the partial region polypeptide of the REIC/Dkk-3 protein ofthe present invention, DNA encoding the polypeptide, and a vectorcontaining the DNA have anticancer immune activity, so that they can beused for: exhibiting therapeutic effects not only on a cancer lesion towhich they are locally administered, but also on cancer metastatic foci;and preventing cancer metastasis.

Furthermore, simultaneous administration of various existing cancerantigen proteins, the partial region polypeptide of the REIC/Dkk-3protein of the present invention, DNA encoding the polypeptide, and avector containing the DNA, can cause systematic anticancer immuneactivation via the induction of differentiation into dendritic(-like)cells, allowing prevention of carcinogenesis itself.

The present invention is hereafter described in detail with reference tothe following examples, although the present invention is not limitedthereto.

Example 1 Preparation of Full-Length Human REIC/Dkk-3 Protein, andPartial Region 1 [Arg121-329] and Partial Region 2 [Gly184-Ile329]Thereof

The full-length [Ala 1-Ile 329] sequence (sample I) (Ala 22 to Ile 350of SEQ ID NO: 2) encoding a mature REIC/Dkk-3 protein and plasmid DNAencoding the REIC partial region [Arg 121-Ile 329] (sample II) of theREIC/Dkk-3 protein were transformed into Escherichia coli (T7 Expressstrain: NEB). About 10 fresh colonies were subjected to mass culture ina 1.6 liter of culture solution. IPTG (0.5 mM) was added to the culturesolution (A600 to 0.7) at the logarithmic growth phase, so as to induceprotein expression. Cells were cultured under expression inductionconditions at 37° C. for 3 hours and then collected by centrifugation.The cells expressing proteins were lysed by ultrasonication or the like,centrifugation was performed, and then the thus expressed proteins werecollected in an insoluble fraction. To remove E. coli cell-derivednucleic acids contaminating the insoluble fraction insofar as possible,10 unit/mL Benzonase (Novagen) was added to the insoluble fractiondispersed in a buffer containing 20 mM Tris-HCl (pH8.0) and 5 mM MgCl₂,incubation was performed at 25° C. for 30 minutes, and thus nucleic aciddegradation was accelerated. Subsequently, centrifugation was performedagain, so that the insoluble fraction containing the high-purityREIC/Dkk-3 protein was obtained. Next, the precipitate thereof wassuspended and dissolved well in Tris-HCl buffer (pH 8) containing 6 Mguanidine hydrochloride, 100 mM 2-mercaptoethanol was added, incubationwas performed at 37° C. for 1 hour, and thus the protein was completelyreduced. At this time, protein quantification was performed by theBradford method to determine the protein concentration. The resultantwas diluted with a refolding buffer so that the final proteinconcentration was 0.2 mg/mL, followed by 24 hours of incubation at 25°C. The buffer (to be used at the time of refolding) containing 20 mMTris-HCl, pH8.0, 0.4 M guanidine hydrochloride, and 30% glycerol, wasprepared in advance to have a composition such that it containedoxidized glutathione (NACALAI TESQUE, INC.) in an amount (in terms ofmoles) ¼ that of 2-mercaptoethanol (contained in the reduced proteinsolution, so that it was introduced into the refolding buffer). Thereduced protein solution was immediately diluted with the buffer havingthe aforementioned composition with stirring well using a stirrer. Afterrefolding under the oxidation-reduction conditions, protein adsorptionwas performed using a column filled with an anion exchange resin(DEAE-Toyopearl 650 M, TOSOH CORPORATION). Elution was performed in 20mM Tris-HCl buffer (pH 8.0) with a linear concentration gradient (0 to0.8 M) of sodium chloride, and then the peak fraction of the REICprotein was collected at a sodium chloride concentration of about 0.5 M.At this time, full-length REIC [Ala 1-Ile 329] (sample I) was collectedin the form of an oligomer with a disulfide (SS) bond formed betweenmolecules. Accordingly, 30 mM dithiothreitol (DTT) was further added andthen incubation was performed at 37° C. for 1 hour, thus resulting in amonomer in which intermolecular S—S bond alone was reduced to a limitedextent. After this limited reduction reaction, the resultant wasimmediately equilibrated with phosphate buffer or MES buffer adjusted tohave pH 6.0. DTT removal and buffer (pH 6.0) exchange were performed bySephadex G25M column chromatography (GE HEALTHCARE), so that a samplethat could be stably stored at 4° C. for several weeks was collected.The sample obtained at this stage is sample I. Sample I could beconcentrated as necessary using a ultrafiltration filter through whichonly substances having a molecular weight of 10 kDa or less can pass.

With the above procedures, the REIC partial region [Arg 121-Ile 329](sample II) was isolated as a stable monomer by anion exchangechromatography. To further perform high-purity purification, elution wasperformed by anion exchange HPLC using a Resource-Q column (GEHEALTHCARE) or the like and a linear concentration gradient of sodiumchloride. Thus, the high-purity REIC partial region [Arg 121-Ile 329] ofthe REIC/Dkk-3 protein could be purified. The resultant was subjected tobuffer exchange by Sephadex G25M column chromatography equilibrated withPBS (pH 7.4), so that sample II was obtained. Sample II could beconcentrated as necessary using a ultrafiltration filter through whichonly substances having a molecular weight of 10 kDa or less can pass.

A protein encoding the partial region [Gly 184-Ile 329] (sample III) ofthe REIC/Dkk-3 protein was prepared as follows. Expression plasmid DNAwas transformed into Escherichia coli (Shuffle T7 Express strain: NEB),and then about 10 fresh colonies were subjected to mass culture in a 0.8liter of culture solution. IPTG (0.5 mM) was added to the culturesolution (A600 to 0.7) at the logarithmic growth phase, so as to induceprotein expression. Cells were cultured under expression inductionconditions at 30° C. for 16 hours and then collected by centrifugation.The cells expressing proteins were lysed by ultrasonication or the like,centrifugation was performed, and then the thus expressed proteins werecollected in a soluble fraction. In the REIC protein [Gly 184-Ile 329]used in this example, a His tag sequence was added to the N-terminalside. Thus, affinity purification was performed using TALON MetalAffinity Resin (Clontech). For further high-purity purification, theresultant was purified by anion exchange HPLC using a Resource-Q column(GE HEALTHCARE) or the like, and then subjected to buffer exchange bySephadex G25M column chromatography equilibrated with PBS (pH 7.4), sothat sample III was obtained.

The molecular weights inferred from the amino acid sequences were: 37.5kDa in the case of the full-length REIC/Dkk-3 protein; 26.9 kDa in thecase of the REIC/Dkk-3 protein partial region 1 [Arg 121-Ile 329], and19.7 kDa in the case of the REIC/Dkk-3 protein partial region 2 [Gly184-Ile 329]. Migration in the form of single bands toward molecularweights as inferred was confirmed by SDS-PAGE analysis.

The amino acid sequence of the full-length REIC/Dkk-3 protein is shownin SEQ ID NO: 2, the amino acid sequence of the REIC/Dkk-3 proteinpartial region 1 [Arg 121-Ile 329] is shown in SEQ ID NO: 5, and theamino acid sequence of the REIC/Dkk-3 protein partial region 2 [Gly184-Ile 329] is shown in SEQ ID NO: 3. Furthermore, the sequencecontaining a His tag added to these sequences is shown in SEQ ID NO: 11.In addition, the amino acid sequence of the REIC/Dkk-3 protein partialregion 1 [Arg 121-Ile 329] corresponds to amino acids 142 to 350 of theamino acid sequence shown in SEQ ID NO: 2.

FIG. 1 shows the outline of a method for preparing the full-length humanREIC/Dkk-3 protein, the partial region 1 [Arg121-329], and the partialregion 2 [Gly184-Ile329] thereof.

Example 2 Induction of Dendritic Cell-Like Differentiation fromPeripheral Blood Mononuclear Cell Preparation of Human Monocyte

Human PBMCs (peripheral blood monocytes) were prepared from the blood ofhealthy donors by a standard method involving Ficoll-Paquecentrifugation. The cell collection rate was determined by the trypanblue exclusion method. The survival rate was confirmed to be 99% orgreater. For preparation of monocytes, PBMCs were resuspended in LGM-3(serum-free lymphocyte growth medium-3; Lonza). The cells adhering to aplastic dish (subjected to incubation in a 10-cm dish at 37° C. for 2hours) were used as monocytes. In some experiments, CD14+ monocytes wereseparated using CD14+ magnetic-activated cell sorting microbeads (MACS;MiltenyiBiotec). Purified CD14+ monocytes were resuspended in LGM-3medium. Using flow cytometry, the purity was always found to exceed 95%.

Treatment of Human Monocytes

PBMCs were cultured alone (no addition), or cultured in the presence ofGM-CSF (R&D Systems)+IL-4 (R&D Systems) (2 ng/ml each), the full-lengthhuman REIC/Dkk-3 protein, the partial region 1 [Arg 121-Ile 329], or thepartial region 2 [Gly 184-Ile 329] (10 μg/ml) thereof prepared inExample 1. The cells were observed with a phase contrast microscope.

FIG. 2 shows the results of inducing dendritic cell-like differentiationon day 7 of culture in PBMCs cultured alone (no addition), or culturedin the presence of GM-CSF (R&D Systems)+IL-4 (R&D Systems) (2 ng/mleach), in the presence of the full-length human REIC/Dkk-3 protein, thepartial region 1 [Arg 121-Ile 329], and the partial region 2 [Gly184-Ile 329] (10 μg/ml) thereof prepared in Example 1. FIG. 2A shows theresult of culturing PBMCs alone, FIG. 2B shows the result of addingGM-CSF+IL-4, FIG. 2C shows the result of adding the full-lengthREIC/Dkk-3 protein, FIG. 2D shows the result of adding the REIC/Dkk-3protein partial region 1 [Arg 121-Ile 329], and FIG. 2E shows the resultof adding the REIC/Dkk-3 protein partial region 2 [Gly184-Ile 329]. FIG.2 shows slightly expanded phase contrast microscopic images.

As a result of morphological observation, the strongest activity ofinducing dendritic-cell-like cell differentiation from peripheral bloodmononuclear cells was observed in the case of the REIC/Dkk-3 proteinpartial region 2 [Gly 184-Ile 329].

FIG. 3 shows the results of comparing differentiation induction in thecase in which PBMCs alone was added, in the case in which GM-CSF+IL-4was added, and in the case in which the REIC/Dkk-3 protein partialregion 2 [Gly 184-Ile 329] was added. FIG. 3A shows the result ofculturing PBMCs alone, FIG. 3B shows the result of adding GM-CSF+IL-4,FIG. 3C shows the result of adding the REIC/Dkk-3 protein partial region2 [Gly 184-Ile 329]. FIG. 3 shows significantly expanded phase contrastmicroscopic images.

Dendritic-cell-like cells resulting from differentiation induction fromperipheral blood mononuclear cells in the case in which the REIC/Dkk-3protein partial region 2 had been added were morphologically smallerthan dendritic cells induced with IL-4+GM-CSF. Meanwhile, nomorphological difference was observed between dendritic-cell-like cellsinduced in the cases in which the full-length REIC/Dkk-3 protein and theREIC/Dkk-3 protein partial region 1 [Arg121-Ile329] had been added anddendritic-cell-like cells induced in the case in which the REIC/Dkk-3protein partial region 2 had been added.

FIG. 4 shows the frequency of the occurrence of dendritic-cell-likecells as a result of culturing PBMCs alone, adding GM-CSF (R&DSystems)+IL-4 (R&D Systems) (2 ng/ml each), adding the full-length humanREIC/Dkk-3 protein, adding the partial region 1 [Arg 121-Ile 329], oradding the partial region 2 [Gly 184-Ile 329] (10 μg/ml). On Day 7, theresultants were stirred manually. Three (3) minutes later, the number ofdendritic-cell-like cells per randomly-selected visual field was countedwith the magnification of the slightly expanded photographs. The thusobtained data were converted into a graph (n=5 visual fields). As aresult of morphological observation, the strongest activity of inducingdendritic-cell-like cell differentiation from peripheral bloodmononuclear cells was observed in the case of the REIC/Dkk-3 proteinpartial region 2 [Gly 184-Ile 329]. The activity in the case in whichthe REIC/Dkk-3 protein partial region 2 [Gly 184-Ile 329] had been addedwas significantly stronger than that in the case in which thefull-length REIC/Dkk-3 protein had been added and that in the case inwhich the partial region 1 [Arg 121-Ile 329] had been added.Furthermore, the activity in the case in which the REIC/Dkk-3 proteinpartial region 2 [Gly 184-Ile 329] had been added was also stronger thanthat in the case in which GM-CSF+IL-4 had been added.

Example 3 Preparation of the Partial Region 3 [Ser114-Phe267] of theHuman REIC/Dkk-3 Protein

As host cells for protein production, human kidney-derived cellsFreeStyle 293-F cells (Invitrogen) at the logarithmic growth phase wereused. Five (5) 500-mL flasks each containing 180 mL of a solution of thehuman kidney-derived cells with a concentration of 5 to 6×10⁵ cells/mLwere prepared. Cells were cultured with shake (125 rpm) over night at37° C. in the presence of 8% CO₂ using Freestyle 293 Expression Media(Invitrogen). On the next day, each solution was adjusted to aconcentration of 1×10⁶ cells/mL. 180 μg each of a high expressionplasmid (described below) encoding the full-length REIC [Ala1-Ile329]was mixed with 293 Fectin (Invitrogen) and then 180 mL of the solutionof 293-F cells added to each 500-mL flask was transiently transfectedwith the mixture. After transfection, shake culture was performed for 4days at 37° C. in the presence of 8% CO₂, and then culture supernatantswere collected.

The thus collected culture supernatants were concentrated byultrafiltration, the solvent was substituted with 20 mM Hepes Buffer(pH7.2) using Sephadex G25M column chromatography (GE HEALTHCARE), andthen REIC protein-containing fractions were collected. Subsequently,protein adsorption was performed by anion exchange column chromatography(DEAE-Toyopearl 650M, TOSOH CORPORATION), and then elution was performedin 20 mM Hepes Buffer (pH7.2) with a linear concentration gradient (0 to0.7M) of sodium chloride. Under conditions of the sodium chlorideconcentration of about 0.35 M, the peak fraction of the REIC protein wasconfirmed. The REIC protein was collected from a fraction containing theREIC protein, the number and the purity of which were higher than thatcontained in the peak fraction.

After buffer exchange with PBS using Sephadex G25M column chromatographyequilibrated with PBS (pH7.4), followed by 5 days of incubation at 37°C. or room temperature, the full-length REIC [Ala1-Ile329] protein wassubjected to limited proteolysis to result in the protein encoding theREIC partial region [Ser 114-Phe 267]. A protein solution containing theREIC partial region [Ser 114-Phe 267] was subjected to solventsubstitution with 20 mM Hepes Buffer (pH7.2) by Sephadex G25M columnchromatography (GE HEALTHCARE), so that a REIC protein-containingfraction was collected. Subsequently, protein adsorption was performedby anion exchange column chromatography (DEAE-Toyopearl 650M, TOSOHCORPORATION), and then elution was performed in 20 mM Hepes Buffer(pH7.2) with a linear concentration gradient (0 to 0.6 M) of sodiumchloride. Under conditions of the sodium chloride concentration of about0.3 M, the peak fraction of the REIC partial region [Ser 114-Phe 267]protein was confirmed. The REIC partial region [Ser 114-Phe 267] proteinwas collected from the peak fraction, so that sample IV was obtained.Sample IV was concentrated as necessary using an ultrafiltration filterthrough which substances having a molecular weight of 10 kDa or less canpass.

FIG. 5-1 shows preparation protocols. Also, FIG. 5-2 shows a chart (FIG.5-1 (3) anion exchange chromatography) showing the result of the 1^(st)purification by anion exchange chromatography. FIG. 5-3 shows a chart(FIG. 5-1(4) anion exchange chromatography) showing the result of the2^(nd) purification by anion exchange chromatography.

The high expression plasmid used in this example is a plasmid containingan expression cassette having a specific structure that enables massproduction of a target protein (to be expressed by gene expression)through ultrahigh expression. The expression cassette has a structure inwhich a DNA construct containing the gene (to be expressed) of a protein(to be expressed) and a polyA addition sequence is located at leastdownstream of the 1^(st) promoter, and an enhancer or the 2^(nd)promoter is ligated downstream of the construct. At the farthest regiondownstream of the expression cassette, the above enhancer or the 2^(nd)promoter is present, and no other gene expression mechanisms are presentdownstream thereof. Specifically, the expression cassette to be used inthe present invention has a structure in which at least a gene to beexpressed is sandwiched between one 1^(st) promoter and at least oneenhancer, or between one 1^(st) promoter and one 2^(nd) promoter. Here,the term “other gene expression mechanisms” refers to a mechanism forexpressing genes other than a gene to be expressed. As a promoter, aCMVi promoter, an SV40 promoter, an hTERT promoter, a β actin promoter,or a CAG promoter can be used. As an enhancer, a CMV enhancer, an SV40enhancer, or an hTERT enhancer can be used. Furthermore, DNA encoding aprotein to be expressed may be ligated downstream of a promoter, and 1to 4 CMV enhancers may be ligated upstream of a DNA construct containinga polyA addition sequence. Moreover, (i) RU5′ ligated immediatelyupstream of DNA encoding a foreign protein, (ii) UAS ligated immediatelyupstream of an enhancer and/or promoter, or (iii) SV40-ori ligated tothe farthest region upstream of an expression cassette may also becontained. FIG. 9 shows the construct of an expression cassettecontained in the plasmid used herein.

Example 4 Induction of Dendritic Cell-Like Differentiation fromPeripheral Blood Mononuclear Cells by the Partial Region 3[Ser114-Phe267] of the Human REIC/Dkk-3 Protein

With the method described in Example 2, human PBMCs (peripheral bloodmonocytes) were prepared and then cultured alone (no addition), culturedin the presence of the full-length human REIC/Dkk-3 protein, cultured inthe presence of the partial region 1 [Arg 121-Ile 329] thereof preparedin Example 1, or cultured in the presence of the partial region 3[Ser114-Phe267] (10 μg/ml) prepared in Example 3. Cells were observedwith a phase-contrast microscope.

FIG. 6 shows the results of inducing dendritic cell-like differentiationon Day 7 of culture in PBMCs cultured alone (no addition), cultured inthe presence of the full-length human REIC/Dkk-3 protein prepared inExample 1, and cultured in the presence of the partial region 3[Ser114-Phe267] (10 μg/ml) prepared in Example 3, respectively. FIG. 6Ashows the result of culturing PBMCs alone, FIG. 6B shows the result ofadding the full-length REIC/Dkk-3 protein, FIG. 6C shows the result ofadding the REIC/Dkk-3 protein partial region 3 [Ser114-Phe267]. FIG. 6shows slightly expanded phase contrast microscopic images. As a resultof morphological observation, in the case of the REIC/Dkk-3 partialregion 3, activity of inducing dendritic-cell-like cell differentiationfrom peripheral blood mononuclear cells equivalent to or higher thanthat in the case of the full-length REIC/Dkk-3 protein or the partialregion 1 thereof was observed. FIG. 7 shows significantly expandedimages. As shown in FIG. 7, no morphological difference was observed indendritic-cell-like cells resulting from differentiation induction fromperipheral blood mononuclear cells between the case of REIC/Dkk-3partial region 3 and the case of partial region 1.

Example 5 Stability of REIC/Dkk-3 Partial Region 3

After 5 days of incubation at 37° C. or room temperature (about 20° C.),18 μL of each sample was separated using SDS-PAGE. The protein encodingthe REIC/Dkk-3 partial region 3 having a molecular weight of about 17kDa was detected by CBB staining. At this time, each sample was left tostand at room temperature (about 20° C.) or 37° C. for 5 days, and thensubjected to SDS-PAGE, so that the degradation pattern was confirmed.FIG. 8 shows the result of SDS-PAGE. Regardless of a temperature thatwas too high for protein storage conditions, the partial region 3 wasdetected by CBB staining as a band of about 17 kDa after SDS-PAGEseparation.

Furthermore, it was demonstrated that in the case of the proteinsolution of the partial region 3, no reactions were observed such asaggregation and precipitation even when salts (e.g., various PEGs,ammonium sulphide, and lithium sulfide) or alcohols (e.g.,2-methyl-2,4-pentandiol (MPD), ethanol, and 2-propanol) had been addedand mixed therewith. Thus, it was revealed that the partial region 3 hadsufficiently high stability.

Example 6 Determination of Tumor-Suppressive Effects of the PartialRegion 3 [Ser 114-Phe 267] of the REIC/Dkk-3 Protein in In VivoExperiments

RENCa cells (1×10⁶) were subcutaneously injected into mice (BALB/c,female, n=5). On Days 3, 5, 7, 10, 12, and 14 after injection (providedthat Day 3 after injection was designated as the day of the start ofadministration of the full-length REIC protein and the partial region3), the full-length REIC protein (100 μg (100 μl)) or the partial region3 (100 μg (100 μl)) or PBS (100 μl) as a control was intraperitoneallyinjected into mice. On Day 17, therapeutic effects in subcutaneoustumors were determined, anticancer immune activity was measured (Example7), and then mice were euthanized. FIG. 10 shows in vivo experimentalprotocols. Tumor volume was obtained by the following formula:0.52×(minimum diameter)²×(maximum diameter).

FIG. 11A shows changes over time in tumor volume after treatment. Thetumor volumes of the group treated with the full-length REIC protein ortreated with the partial region 3 and the group to which PBS as acontrol had been administered were compared. As a result, differenceswere found to be statistically significantly small (indicated with *).FIG. 11B shows the weights of tumors collected from mice. The tumorweights of the group treated with the full-length REIC protein or thepartial region 3 and the group to which PBS had been administered werecompared. As a result, differences were found to be statisticallysignificantly small (indicated with *). FIG. 11C shows photographs oftumors collected from mice. As shown in FIG. 11A to FIG. 11C, tumorgrowth could be suppressed through administration of the full-lengthREIC protein and the partial region 3.

Example 7 Flow Cytometry of the Immunocompetent Cells in Mouse VenousBlood of Example 6

Changes in the number of the immunocompetent cells existing in mousevenous blood obtained in Example 6 were analyzed by flow cytometry. 0.2%EDTA solution (30 μl) was added to 750 μl of mouse blood collected frominferior vena cava for anticoagulation. Each of the following antibodies(1 μl each) fluorescently labeled differently (purchased fromeBioscience) was added to 30 μl of blood. The resultants were stirredand then incubated at 4° C. for 60 minutes, so that the followingimmunocompetent cells were stained.

Bone marrow-derived immunosuppression cells (anti-GR-1 antibody,anti-CD11b antibody)Dendritic cells (anti-CD11 antibody)Activated dendritic cells (CD11c+/CD80+) (anti-CD 11c antibody,anti-CD80 antibody)Activated dendritic cells (CD11c+/CD86+) (anti-CD11c antibody, anti-CD86antibody)Helper T cells (anti-CD4 antibody)Immunoregulatory T cells (anti-CD4 antibody, anti-Foxp3 antibody)Cytotoxic T cells (anti-CD8 antibody)Activated cytotoxic T cells (anti-CD69 antibody, anti-CD8 antibody)NK cells (anti-CD3e antibody, anti-NK1.1 antibody)

Subsequently, erythrocytes were lysed in a red blood cell lysis buffer.Cells were washed twice with PBS, suspended again in 200 μl of PBS, andthus a cell solution to be analyzed was prepared. 3×10⁴ cells werecollected using a FACS Calibur flow cytometer (Becton Dickinson) andthen analyzed using CellQuest software (Becton Dickinson). Anappropriate gate was set on the basis of the forward scatter patterncharacteristic of these cells, and thus only cells within the gate wereanalyzed. As a result, the full-length REIC protein and the partialregion 3 were observed to exhibit activity of inducing differentiationto almost all immunopotentiating cells (FIG. 12B, C, D, E, G, H, I)while also observed to exhibit activity of suppressing thedifferentiation induction of immunosuppressive cells (FIG. 12A, F). Inparticular, the partial region 3 was excellent in induction of cytotoxicT cell (CTL) differentiation (FIG. 12G, H) and was observed to exhibitstrong activity of suppressing immunoregulatory T cell (Treg)differentiation (FIG. 12F). As described above, it can be concluded thata protein containing the partial region 3 is applicable as an anticancerimmunopotentiating agent, an anticancer agent, an antitumor agent, or anagent for inducting/suppressing immune cell differentiation. The sameapplies to the full-length REIC protein containing the partial region 3.

Example 8 Experiment Concerning the Anti-Tumor Effects of theFull-Length REIC Protein Using Renal Cell Carcinoma Model Mice

Mouse renal cell carcinoma cells (RENCA-Luc cell line) were prepared bycausing a RENCA cell line to stably express a Luciferase gene. Toexamine the tumor suppressive effects of the REIC protein by an in vivoexperiment, renal cell carcinoma model mice were produced using theRENCA-Luc cell line. For preparation of orthotopic tumors, male BALB/Cmice were anesthetized with Nembutal and then RENCA-Luc cells (10³cells) were locally injected into the left kidney. Immediately afterinjection, 10⁴ cells were injected via tail vein, so that a renal cellcarcinoma mouse model having pulmonary metastatic foci was prepared (Day0). A control group and a treatment group were treated as follows.

A: PBS (100 μl) was intraperitoneally administered every day (13 days) atotal of 13 times.B: REIC protein (10 μg/100 μl PBS) was intraperitoneally administeredevery day (13 days) a total of 13 times.C: REIC protein (100 μg/100 μl PBS) was intraperitoneally administeredevery day (13 days) a total of 13 times.

On Day 14 after the start of administration (Day 14), mice wereeuthanized. The thus excised tumors were analyzed for tumor size andtumor weight. As shown in FIG. 14A, tumor shrinkage was observed in thegroup treated with the REIC protein, compared with the group ofuntreated mice and the group treated with the buffer. It was suggestedthat the tumor shrinkage effect became more significant depending on thedose of the REIC protein (FIG. 14B). Tumor shrinkage was also similarlyobserved in pulmonary metastatic foci (FIG. 15A, B).

Example 9 Differentiation Induction Experiment Using Mouse Bone MarrowCells

Bone marrow was collected from an untreated normal mouse and thensuspended by pipetting. Mouse bone marrow cells were cultured in a flatbottom 6-well plate. On the next day, cells were washed twice with PBS,and then cells that had adhered were used for the experiment. Throughthe addition of GM-CSF (20 ng/ml, purchased from R&D Systems) alone orthe addition of GM-CSF and the full-length REIC protein (10 μg/ml), theeffects of the REIC protein to suppress the induction of thedifferentiation to myeloid derived suppressor cells (MDSC) wereanalyzed. On Day 6 after the start of the culture of mouse bone marrowcells, cells were collected by trypsin treatment and then stained withantibodies against surface antigen markers (GR-1, CD11b) of MDSC. 3×10⁴cells were analyzed using a FACS Calibur flow cytometer (BectonDickinson). Whereas the percentage of MDSC was 2.56% in the case of notreatment, the percentage of MDSC increased to 53.04% in the case inwhich GM-CSF had been administered (FIG. 16A, B). This means that GM-CSFenhanced the induction of MDSC differentiation, so as to suppress thecapacity of immune activation. When GM-CSF and the full-length REICprotein were mixed, differentiation to MDSC was induced at a rate(34.71%) lower than the case of GM-CSF alone. The result means that theREIC protein suppressively acts on induction of MDSC differentiation, soas to exhibit the capacity of immune activation.

Example 10 Flow Cytometry of the Immunocompetent Cells in Mouse VenousBlood of Example 8

The percentage of the immunocompetent cells in mouse venous bloodobtained in Example 8 was analyzed by flow cytometry. In a mannersimilar to Example 7, 30 μl of a 0.2% EDTA solution was added to 750 μlof mouse peripheral blood collected from inferior vena cava foranticoagulation. Each of the following antibodies (1 μl each)fluorescently labeled differently (purchased from eBioscience) was addedto 30 μl of blood, the solution was stirred, incubation was performed at4° C. for 60 minutes, and thus each type of the followingimmunocompetent cells was stained.

Bone marrow-derived immunosuppression cells (anti-GR-1 antibody,anti-CD11b antibody)Activated dendritic cells (CD11c+/CD80+) (anti-CD11c antibody, anti-CD80antibody)Immunoregulatory T cells (anti-CD4 antibody, anti-Foxp3 antibody)Activated cytotoxic T cells (anti-CD69 antibody, anti-CD8 antibody)NK cells (anti-CD3 antibody, anti-NK1.1 antibody)

Subsequently, erythrocytes were lysed in a red blood cell lysis buffer.Cells were washed twice with PBS, suspended again in 200 μl of PBS, andthus a cell solution to be analyzed was prepared. 3×10⁴ cells werecollected using a FACS Calibur flow cytometer (Becton Dickinson) andthen analyzed using CellQuest software (Becton Dickinson). Anappropriate gate was set on the basis of the forward scatter patterncharacteristic of these cells, and thus only cells within the gate wereanalyzed. FIG. 17A to FIG. 17E show the results. In the cases ofdendritic cells (FIG. 17B-1 and B-2), activated cytotoxic T cells (CTL,FIG. 17D-1 and D-2) and NK cells (FIG. 17E-1 and E-2), the positive ratewas found to increase depending on the dose of the REIC protein. All ofthese immunocompetent cells function to accelerate immunoactivity.Moreover, it was demonstrated that of MDSC (FIG. 17A-1 and A-2) and Treg(FIG. 17C-1 and C-2) suppressively act on the immune system, thepositive rate decreases depending on the dose of the REIC protein.

As described above, it was found that the REIC protein has the functionof activating the immune system by attenuating the immunosuppressionsystem. Decreased immunocompetence in an in vivo micro environment isknown to be able to accelerate tumor development and growth, which ismainly caused by the development and induction of immunosuppressivecells. Therefore, the REIC protein and a DNA vector expressing theprotein are applicable as agents for inhibiting immunosuppression, suchas anticancer agents, antitumor agents, agents for activating anticancerimmunity, and agents for inducing immunocompetent cell differentiation.

Example 11 Analysis of Interaction Between REIC Protein and Tctex-1 byYeast 2-Hybrid Method (Y2H)

The ProQuest Two-hybrid System (Invitrogen, Carlsbad, Calif.) was usedfor the yeast 2-hybrid method. The full-length cDNA of human REIC/Dkk-3was amplified by the following two primer DNAs.

Forward: (SEQ ID NO: 12) 5′-ACGCGTCGACCATGCAGCGGCTTGGGGCCAC-3′ Reverse:(SEQ ID NO: 13) 5′-TTCCTTTTTTGCGGCCGCTAAATCTCTTCCCCTCCCA-3′

The thus amplified cDNA was inserted between Sal 1 and Not 1 enzymecleavage sites of a pDBLeu bait vector, and then the vector wasintroduced into the yeast MaV203 strain. Subsequently, a REIC/Dkk-3expressing clone was isolated and then transformed into the human heartcDNA library cloned into pPC86 (Invitrogen). Clones that had beensuccessfully introduced were collected using selective mediumsupplemented with β-galactosidase substrate. Gene introduction, plasmidisolation, confirmation of DNA constructs, and preparation of yeastlysates were performed referring to the instructions of Invitrogen.

To identify interaction partners for REIC/Dkk-3, the yeast 2-hybridmethod screening was performed using the REIC/Dkk-3 protein as a bait.REIC/Dkk-3 expression is enhanced in mouse and human cardiac tissue, ahuman cDNA library of normal cardiac tissue was subjected to screening.Among clones with activated reporter genes, 4 clones were successfullycultured. Inserted genes were separated and collected from these clonesby a plasmid rescue method, gene introduction was performed again, andthen culture was performed using reporter selective media. FIG. 18Ashows the results. In FIG. 18A, blue colonies were observed in the rightpanel containing Tctex-1. Blue colonies clearly indicate the interaction(association) between the REIC/Dkk-3 protein and the Tctex-1 protein.These results suggested that Tctex-1 is an interaction partner forREIC/Dkk-3 (FIG. 18A).

Example 12 Analysis of REIC-Tctex-1 Interaction by Immunoprecipitationand Western Blot

For the purpose of confirming binding partners by theimmunoprecipitation method, the human full-length cDNA of REIC/Dkk-3 orTctex-1 was inserted into a pcDNA3.1/Myc-His(−)A or apcDNA3.2/V5/GW/D-TOPO plasmid (Invitrogen), followed by cloning. For thepurpose of transient gene expression, 293T cells were transfected withthe plasmid DNA using lipofectamine 2000. 293T cells were co-transfectedwith Myc-tagged REIC/Dkk-3 or V5-tagged Tctex-1. At 48 hours aftertransfection, 293T cells were lysed, a buffer (20 mM Tris-HCl, pH 7.5,1% Triton X-100, 150 mM NaCl, 5 mM EDTA and Complete Protease InhibitorCocktail (Roche, Basel, Switzerland)) was added. One (1) mg of amouse-derived non-specific IgG antibody (Santa Cruz Biotechnology, SantaCruz, CA) was added to the cell lysate, incubation was performed, andthen 1 ml of protein G sepharose (Invitrogen) or an anti-Mycmouse-derived monoclonal antibody (Invitrogen, Cat. No. R95025) wasadded. After 12 hours of incubation at 4° C., the precipitates werewashed and then boiled in a buffer in which SDS had been dissolved. Theprecipitates were subjected to the Western blot method using amouse-derived V5 monoclonal antibody (Invitrogen, Cat. No. R96025).

To confirm the interaction between REIC/Dkk-3 and Tctex-1, in vitro pulldown assay was performed by the immunoprecipitation method. Bindingassay of the V5-Tctex-1 fusion protein was performed using a cell lysateof 293T cells expressing the Myc-REIC/Dkk-3 fusion protein. Binding toTctex-1 was detected by the Western blot method using a V5 antibody.Binding of Tctex-1 to the REIC/Dkk3 protein was detected from animmunoprecipitation sample obtained by adding the Myc antibody to celllysates of cells co-transfected with REIC/Dkk-3 and Tctex-1. FIG. 18Bshows the results. The results of Examples 11 and 12 demonstrate thatthe interaction between the REIC/Dkk-3 protein and the Tctex-1 proteinwas confirmed to take place and was reproduced by both the yeast2-hybrid method and the immunoprecipitation method.

Example 13 Analysis of the Region of the REIC Protein Binding to Tctec-1by the Mammalian 2-Hybrid Method (M2H)

To perform the mammalian cell 2-hybrid assay, cDNAs encoding REIC/Dkk-3with various amino acid lengths were introduced into plasmids forcloning a pM GAL4 DNA binding domain. Furthermore, cDNA encodingfull-length Tctex-1 was introduced into a plasmid for cloning a pVP16transcriptional activity domain (Clontech Laboratories, Mountain View,Calif.). Templates for human REIC/Dkk-3 each having different amino acidlengths were generated and amplified by the PCR method using appropriateprimer pairs. The complete full-length cDNA of Tctex-1 was amplifiedwith the following primers.

Forward (SEQ ID NO: 14) 5′-CCGGAATTCATGGAAGACTACCAGGCTGC-3′ Reverse(SEQ ID NO: 15) 5′-GGGAAGCTTTCAAATAGACAGTCCGAAGG-3

About 2×10⁵ 293T cells were co-transfected with 400 ng of pVP16, 400 ngof pM, 250 ng of pFR-Luc firefly-derived luciferase reporter plasmid(Promega, Madison, Wis.), and 10 ng of phRL-TKRenilla-derived luciferasereporter plasmid (Promega). The transfected cells were cultured for 48hours, and then luciferase activity was measured using a dual-luciferasereporter assay system (Promega). Errors due to differences intransfection efficiency were normalized by measuring Cypridina-derivedluciferase activity resulting from the transfection of phRL-TK gene.

Interaction between REIC/Dkk-3 and Tctex-1 was analyzed by the mammalian2-hybrid method for searching for REIC/Dkk-3 partial regions importantfor interaction with Tctex-1. 293T cells were co-transfected with a GAL4plasmid with REIC/Dkk-3 cDNA (of each amino acid length) introducedthereinto and a VP 16 plasmid with the cDNA of full-length Tctex-1introduced thereinto. Luciferase activity in the cell lysate of 293Tcells was measured. When the activity was observed, binding of eachREIC/Dkk-3 partial region to Tctex-1 was determined. As a result, it wasrevealed that a REIC/Dkk-3 partial region composed of 20 to 146 aminoacid residues is important as a binding region binding to Tctex-1.Furthermore, it was revealed that REIC/Dkk-3 partial regions havingamino acid lengths longer than that of the aforementioned partial regionhave low activity of binding to Tctex-1 (FIG. 19A). To further selectthe regions of REIC/Dkk-3 for binding to Tctex-1, REIC/Dkk-3 partialregions were shortened in stages. Luciferase activity indicating thebinding with Tctex-1 was detected in REIC/Dkk-3 having 20-146 aminoacids and partial regions of 20-157 amino acid residues. Only weakactivity was observed for a partial region composed of 20-135 amino acidresidues. These results suggest that a REIC/Dkk-3 partial regioncomposed of 136-157 amino acid residues is involved in the interactionbetween REIC/Dkk-3 and Tctex-1 (FIG. 19B).

FIG. 20A shows the amino acid sequence alignment of the REIC protein andthe TcTex-1 binding region of a dynein intermediate chain (DIC). Anothergroup has reported the binding of Tctex-1 (that is, a Dynein light chainprotein) with the partial region [¹²⁰ SDSELGRRLHKLGVSKVTQVDFL ¹⁴²] (SEQID NO: 16) of the dynein intermediate chain (DIC). The Tctex-1 bindingregion of REIC/Dkk-3 was found to be [¹³⁶ VGDEEGRRSHECIIDEDCGPSM ¹⁵⁷](SEQ ID NO: 17). Sequence comparison with the Tctex-1 binding region ofDIC revealed that the consensus sequence was [-E-X-G-R-R-X-H-] (Xdenotes an arbitrary natural amino acid) (SEQ ID NO: 18).

FIG. 20B shows the amino acid sequence alignment of the Tctex-1 bindingdomain of the REIC protein and a known Tctex-1 binding protein. Theamino acid sequence motif of the Tctex-1 binding protein was[-R/K-R/K-X-X-R/K-] (X denotes an arbitrary natural amino acid) (SEQ IDNO: 20). The amino acid sequence of the REIC/Dkk-3 binding region isconsistent with other sequences in terms of the motif alone containing[-RR-]. This is characteristic unlike the motifs of other bindingpartners for Tctex-1.

Example 14 Analysis of the Intracellular Localization Patterns ofREIC/Dkk-3 Protein and Tctex-1

Immunocytochemical staining of REIC/Dkk-3 and Tctex-1 in OUMS24 cellswas performed by co-staining the endoplasmic reticulum organelle. Cellswere cultured on 24-well plates under 30% to 40% confluent conditions.Furthermore, cells were fixed with 4% paraformaldehyde. 100 mM phosphatebuffer and then blocked with saline and 3% BSA. A rabbit-derivedanti-REIC/Dkk-3 polyclonal antibody (1:200 dilution in PBS) or arabbit-derived anti-Tctex-1 polyclonal antibody (1:100 dilution in PBS,Santa Cruz Biotechnology, sc-28537) was added, and then cells wereincubated at room temperature for 2 hours. Furthermore, an Alexa488green-fluorescent-dye-conjugated rabbit-derived secondary antibody wasadded, followed by 1 hour of incubation. To detect distribution in theendoplasmic reticulum, Alexa546 red-fluorescent-dye-conjugatedconcanavalin A (Molecular Probes) was added to cells, followed by 15minutes of incubation at room temperature.

In recognition of interaction between REIC/Dkk-3 and Tctex-1 revealed byvarious assay systems, co-localization of the 2 proteins within cellswas analyzed. It has been reported that REIC/Dkk-3 is localized not onlyin the endoplasmic reticulum, but also in the cytoplasm. Our recentstudies have demonstrated that in cells stably expressing the REIC/Dkk-3protein, the REIC/Dkk-3 protein is localized in the endoplasmicreticulum. Accordingly, to confirm localization of the REIC/Dkk-3protein and the Tctex-1 protein in the endoplasmic reticulum, animmunofluorescence double staining method was performed usingconcanavalin A (that is, a fluorescent dye-conjugated endoplasmicreticulum localized marker protein). FIG. 21A shows confocal microscopicimages showing the results of double fluorescent staining of the humanfull-length REIC protein with endoplasmic reticulum marker concanavalinA in OUMS24 human fibroblasts. Red (bright portion in FIG. 21A-a)indicates concanavalin A and green (bright portion in FIG. 21A-b)indicates the full-length REIC protein. The overlay region of theendoplasmic reticulum and the full-length REIC protein is indicated inyellow (bright portion in FIG. 21A-c) (overlay image). FIG. 21B showsconfocal microscopic images showing the results of double fluorescentstaining of the Tctex-1 protein with endoplasmic reticulum markerconcanavalin A in OUMS24 human fibroblasts. Red (bright portion in FIG.21B-a) indicates concanavalin A and green (bright portion in FIG. 21B-b)indicates the Tctex-1 protein. The overlay region of the endoplasmicreticulum and the Tctex-1 protein is indicated in yellow (bright portionin FIG. 21A-c) (overlay image). As shown in overlay images in FIG. 21Aand FIG. 21B, most portions were stained with yellow. Therefore, asexpected, REIC/Dkk-3 (FIG. 21A) and Tctex-1 (FIG. 21B) were bothlocalized in the endoplasmic reticulum and the intracellularlocalization patterns of these proteins in OUMS24 normal fibroblastswere consistent with each other. Specifically, it can be concluded thatboth REIC/Dkk-3 and Tctex-1 are co-localized around the endoplasmicreticulum and that an interaction partner of REIC/Dkk-3 is Tctex-1.

Example 15 Analysis of the Function of Tctex-1 as a Factor to Acceleratethe Capacity of REIC to Induce apoptosis

Adenovirus REIC/Dkk-3 (Ad-REIC) was prepared as follows. A full-lengthcDNA of REIC/Dkk-3 was inserted into a pAxCAwt cosmid vector and thenthe vector was introduced into an adenovirus vector by the COS-TPCmethod (Takara Bio Inc., Shiga, Japan). An adenovirus vector (Ad-LacZ)into which a LacZ gene had been introduced was used as a control.

An apoptosis assay method is as described below. To examine the rate ofin vitro apoptosis induction after each treatment, PC3 prostate cancercells were cultured in flat-bottom 6-well culture plates for 24 hours.The cultured PC3 cells were treated with a GFP-expression plasmid(Clonetech), a Tctex-1-expression plasmid (pcDNA3.2/V5/GW/D-TOPO), or aTctex-1-sh-RNA plasmid (sc-43319-SH, Santa Cruz Biotechnology) addedthereto for 6 hours and then media were exchanged with fresh media.FuGENE HD (Roche) was used for the transfection. The transfectionefficiency of the GFP plasmid was 60% or more at 48 hours after additionof the plasmid. At 24 hours after transfection with the GFP expressionplasmid, Ad-LacZ and Ad-REIC were added at 50 MOI (multiplicity ofinfection) in serum free medium for 2 hours of reaction, and then themedium was exchanged with fresh medium. After 48 hours of culture, a 2μg/ml Hoechst 33342 solution was added to the medium, and then it wasleft to stand in the dark for 10 minutes. Hoechst 33342 is anintercalating reagent for evaluation of total chromatin content anddetection of the degree of chromatin condensation. Highly condensed orfragmented cell nuclei were observed by fluorescence microscopy, anddead cells (apoptotic cells for which apoptosis induction had occurred)were identified. Apoptotic cells were counted by microscopic observationperformed for 5 different fields. 100 cells were counted per microscopicimage.

Statistical analyses (statistical tests) were conducted as describedbelow. Data are shown as the mean±SE. Student's unpaired t-test wasconducted for statistical analyses between the two groups. Differences(between the two) with P values of less than 0.05 were considered to bestatistically significant.

To examine the functional effects of Tctex-1 on REIC/Dkk-3 apoptoticactivity (the capacity to induce apoptosis), a model system (establishedby the present inventors through their previous studies) of apoptosisinduction by REIC/Dkk-3 overexpression using Ad-REIC in PC3 humanprostate cancer cells was used. According to the results of Western blotanalyses, PC3 expressed endogenous Tctex-1 protein but did not expressthe REIC/Dkk-3 protein. FIG. 22A shows the results of apoptosis assayusing Hoechst 33342. In the results of the assay, apoptosis took placein PC3 to which Ad-REIC had been administered, but apoptosis did nottake place in cells treated with Ad-LacZ (indicated with arrows in FIG.22A). FIG. 22A shows that the capacity of Ad-REIC to induce apoptosiswas decreased by a decreased Tctex-1 expression level, but was enhancedby amplified expression thereof. The apoptosis incidences in the groupstreated with Ad-REIC were, specifically, 30%, 15%, and 75%,respectively, in the group treated with the GFP plasmid, the grouptreated with shRNA-Tctex-1, and the group treated with the Tctex-1expression plasmid (FIG. 22B). Specifically, this suggests that thecapacity of Ad-REIC to induce apoptosis is reduced by a decrease inTctex-1 expression level, but is enhanced by amplified expression. Itwas revealed that compared with the group treated with the GFP plasmidto which Ad-REIC had been administered, the group treated withshRNA-Tctex-1 exhibited a decreased apoptosis incidence. Meanwhile, itwas demonstrated that the Tctex-1 expression plasmid acceleratesapoptosis induction. The results suggest that the Tctex-1 expressionlevel is positively correlated with apoptosis induction by REIC/Dkk-3overexpression using Ad-REIC. Therefore, it can be concluded thatTctex-1 accelerates apoptosis induction by REIC/Dkk-3.

INDUSTRIAL APPLICABILITY

The partial region polypeptide of the REIC/Dkk-3 protein of the presentinvention has the following effects, compared with IL-2, a cytokinegroup having many conventionally known immunoactivation effects, and thefull-length REIC/Dkk-3 protein.

(1) The partial region polypeptide has anti-tumor effects even in thesingle-agent form through anticancer immune activation.(2) Because of the REIC/Dkk-3 protein's own property such that manytypes of cancer lack REIC/Dkk-3 gene expression or exhibit REIC/Dkk-3gene expression at low levels, a REIC/Dkk-3 protein preparation iseffective for wide-ranging types of cancer.(3) With anticancer immune activation by the REIC/Dkk-3 protein, effectsof improving or preventing cancer not only at cancer lesions to which itis administered, but also cancer metastatic foci, can be expected.(4) This agent is administered simultaneously with various existingcancer antigen proteins, so that anticancer immunity can besystematically activated via induction of differentiation todendritic(-like) cells and prevention of carcinogenesis itself becomespossible.

Moreover, the size of the partial region polypeptide of the REIC/Dkk-3protein of the present invention is small, so that it can be easilyproduced at low cost.

The REIC/Dkk-3 protein partial region of the present invention can beused as a cancer immunotherapeutic agent and is useful for cancerprevention and treatment.

Sequence Listing Free Text

SEQ ID NO: 11 synthesisSEQ ID NOS: 12-15 primersSEQ ID NOS: 16-28 synthesis

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

1. A polypeptide that is any one of the following polypeptides (a) to (e), which consists of a partial region of a REIC/Dkk-3 protein: (a) a polypeptide consisting of a partial region of the REIC/Dkk-3 protein, which consists of an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, and the amino acid sequence of a polypeptide that contains the partial region consisting of Gly at position 205 to Phe at position 288 of the amino acid sequence shown in SEQ ID NO: 2, and consists of a fragment of the partial region consisting of Ser at position 135 to Ile at position 350 of the amino acid sequence shown in SEQ ID NO: 2; (b) a polypeptide having activity of inducing dendritic-cell-like cell differentiation from monocytes, which is a polypeptide protein consisting of an amino acid sequence that has a substitution, a deletion, or an addition of 1 or several amino acids with respect to an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, and the amino acid sequence of a polypeptide that contains the partial region consisting of Gly at position 205 to Phe at position 288 of the amino acid sequence shown in SEQ ID NO: 2, and consists of a fragment of the partial region consisting of Ser at position 135 to Ile at position 350 of the amino acid sequence shown in SEQ ID NO: 2; (c) a polypeptide consisting of a partial region of the REIC/Dkk-3 protein, which can bind to a Tctex-1 protein and consists of the amino acid sequence shown in SEQ ID NO: 17, and; (d) a polypeptide consisting of an amino acid sequence having a substitution, a deletion, or an addition of 1 or several amino acids with respect to the amino acid sequence shown in SEQ ID NO: 17 and having the activity of binding to the Tctex-1 protein; and (e) a polypeptide consisting of a partial region of the REIC/Dkk-3 protein and being capable of binding to the Tctex-1 protein, which contains the consensus sequence shown in SEQ ID NO: 18 and consists of 7 to 22 amino acid residues.
 2. DNA that is any one of the following DNAs (f) to (l), which encodes a polypeptide consisting of a partial region of a REIC/Dkk-3 protein; (f) DNA consisting of a nucleotide sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, and a nucleotide sequence that contains the partial sequence consisting of g at position 613 to c at position 864 of the nucleotide sequence shown in SEQ ID NO: 1, and consists of a fragment of the partial sequence consisting of t at position 403 to t at position 1050 of the nucleotide sequence shown in SEQ ID NO: 1; (g) DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to a nucleotide sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, and a nucleotide sequence that contains the partial sequence consisting of g at position 613 to c at position 864 of the nucleotide sequence shown in SEQ ID NO: 1, and consists of a fragment of the partial sequence consisting of t at position 403 to t at position 1050 of the nucleotide sequence shown in SEQ ID NO: 1, and encoding a polypeptide having activity of inducing dendritic-cell-like cell differentiation from monocytes; (h) DNA consisting of a nucleotide sequence having a deletion, a substitution, or an addition of 1 or several nucleotides with respect to a nucleotide sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, and a nucleotide sequence that contains the partial sequence consisting of g at position 613 to c at position 864 of the nucleotide sequence shown in SEQ ID NO: 1, and consists of a fragment of the partial sequence consisting of t at position 403 to t at position 1050 of the nucleotide sequence shown in SEQ ID NO: 1, and encoding a polypeptide having activity of inducing dendritic-cell-like cell differentiation from monocytes; (i) DNA consisting of a DNA sequence that encodes an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, and the amino acid sequence of a polypeptide that contains the partial region consisting of Gly at position 205 to Phe at position 288 of the amino acid sequence shown in SEQ ID NO: 2, and consists of a fragment of the partial region consisting of the Ser at position 135 to Ile at position 350 of the amino acid sequence shown in SEQ ID NO: 2, and encoding a polypeptide having activity of inducing dendritic-cell-like cell differentiation from monocytes; (j) DNA encoding a polypeptide that consists of a partial region of the REIC/Dkk-3 protein and is capable of binding to the Tctex-1 protein, which consists of the nucleotide sequence consisting of g at position 4 to g at position 69 of the nucleotide sequence shown in SEQ ID NO: 8; (k) DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence consisting of g at position 4 to g at position 69 of the nucleotide sequence shown in SEQ ID NO: 8, and encoding a polypeptide having activity of binding to the Tctex-1 protein; and (l) DNA encoding a polypeptide that consists of a partial region of the REIC/Dkk-3 protein, is capable of binding to the Tctex-1 protein, consists of the DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 17, and has activity of binding to the Tctex-1 protein.
 3. A vector, containing the DNA of claim
 2. 4. The vector according to claim 3, wherein the vector is an adenovirus vector.
 5. An agent for inducing dendritic-cell-like cell differentiation from monocytes, containing the polypeptide of claim 1 consisting of a partial region of the REIC/Dkk-3 protein as an active ingredient.
 6. An agent for accelerating the induction of differentiation to immunoactivation cells selected from the group consisting of dendritic cells, helper T cells, CTL, and NK cells, containing the polypeptide of claim 1 consisting of a partial region of the REIC/Dkk-3 protein as an active ingredient.
 7. An agent for inhibiting the induction of differentiation to immunosuppressively functioning cells that are myeloid-derived suppressor cells (MDSC) or immunoregulatory T cells (Treg), containing the polypeptide of claim 1 consisting of a partial region of the REIC/Dkk-3 protein as an active ingredient.
 8. An anticancer agent, containing the polypeptide of claim 1 consisting of a partial region of the REIC/Dkk-3 protein as an active ingredient.
 9. An agent for inducing dendritic-cell-like cell differentiation from monocytes, containing the DNA of claim 2 that encodes the polypeptide consisting of a partial region of the REIC/Dkk-3 protein or the vector of claim 3 as an active ingredient.
 10. An agent for accelerating the induction of differentiation to immunoactivation cells selected from the group consisting of dendritic cells, helper T cells, CTL, and NK cells, containing the DNA of claim 2 encoding the polypeptide consisting of a partial region of the REIC/Dkk-3 protein or the vector of claim 3 as an active ingredient.
 11. An agent for inhibiting the induction of differentiation to immunosuppressive cells that are myeloid-derived suppressor cells (MDSC) or immunoregulatory T cells (Treg), containing the DNA of claim 2 encoding the polypeptide consisting of a partial region of the REIC/Dkk-3 protein or the vector of claim 3 as an active ingredient.
 12. An anticancer agent, containing the DNA of claim 2 encoding the polypeptide consisting of a partial region of the REIC/Dkk-3 protein or the vector of claim 3 as an active ingredient.
 13. A method for inducing dendritic-cell-like cell differentiation from CD14 positive monocytes, comprising culturing monocytes collected from an animal in vitro in the presence of the polypeptide of claim 1 consisting of a partial region of the REIC/Dkk-3 protein.
 14. The method according to claim 13, wherein the monocytes are peripheral blood monocytes. 